Antimicrobial hevamine a-related compositions and methods

ABSTRACT

In one aspect, nutraceutical compositions, methods of preparation and methods of use comprise a nutraceutical composition comprising an antimicrobial hevamine A-related protein from Momordica balsamina alone or in combination with one or more nutraceutical ingredients. In another aspect, a method of preventing or treating a microbial infection in a plant comprises applying an effective amount of a composition containing the hevamine A-related protein to a whole plant, plant part, or media in which the plant is growing. In a further aspect, the present application provides a transgenic plant stably transformed with a polynucleotide encoding the hevamine A-related protein.

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 63/208,698, filed on Jun. 9, 2021, the contents of which areexpressly incorporated by reference herein.

FIELD

The present application generally relates to a hevamine A-relatedprotein and methods of use thereof. More particularly, the presentapplication relates to the use of the hevamine A-related protein innutraceutical compositions for a subject. The present application alsodiscloses methods for preventing or treating microbial infections inplants, and transgenic plants.

BACKGROUND

The surfaces of host cells and microorganisms are decorated by complexglycans, which play multifaceted roles in the dynamic interplay betweenthe microorganisms and the host. Such lectins are known to facilitatemicrobial infections by specific multivalent interactions between cellsurfaces decorated by complex glycans and their cognate protein lectins(see e.g., Raman, R. et al., Curr. Opin. Struct. Biol., 40: 153-162,2016).

Lectin proteins are sugar-binding proteins that bind specifically andreversibly to carbohydrate groups. They are typically anchored on thesurfaces of cells and are found in all groups of living organismsincluding plants, animals, fungi, and bacteria, as well as viruses andmycoplasmas. Depending on their broad sugar-binding specificity, theyhave been classified as mannose-, galactose-, N-acetyl-glucosamine-,fucose- and sialic acid-binding lectins, according to the simple sugarsthat inhibit their carbohydrate-binding properties.

The complex glycans displayed on host cell surfaces typically functionas attachment factors, co-receptors or primary receptors that arespecifically recognized by microbial surface glycoprotein similarlydecorated by a variety of glycans. For example, complex glycansterminated by α2-3 or α2-6-linked sialic acid (N-acetyl neuraminic acid)function as receptors for several different viruses. Linear sulfatedglycosaminoglycans such as heparan sulfate act as co-receptors for avariety of viruses, including dengue virus, hepatitis C virus, andfoot-and-mouth disease virus. The display of specific glycan motifs onsurfaces of different cells and tissues contributes to the hostrestriction and cell/tissue tropism of microorganisms.

A wide variety of lectins from animals, plants, algae, cyanobacteria,and other sources have been shown to possess antimicrobial activityagainst a wide variety of bacteria and fungi. Such lectins are alsofound on the surfaces of viruses, including coronaviruses, humanimmunodeficiency viruses (HIVs), influenza viruses, herpes simplexviruses, Ebola viruses, and others. See e.g., Mani et al., Virus Res.,Apr. 30, 2020, pp. 197989; Akkouh et al., Molecules, 20:648-668, 2015).For example, the influenza virus hemagglutinin is one of the mostwell-studied examples of a viral glycan-binding protein and is known tobind to sialic acid-containing glycans on the host cell surface.Additionally, mannose binding lectin (MBL), a serum protein in humansimportant in host defenses has been shown to selectively bind to theSARS CoV Spike (S) protein in a SARS-CoV pseudotyped virus and potentlyinhibit SARS-CoV infection of susceptible cell lines at concentrationsbelow those observed in the serum of healthy individuals (Zhou, Y etal., J. Virol., 84(17): 8753-8764, 2010). Exemplary lectins with broadspectrum antiviral activity against multiple viruses includeConcanavalin A from jack bean, Griffithsin from red algae, andCyanovirin-N from cyanobacteria.

Similarly, lectins mediate adhesion of bacteria to host cells ortissues, which is a prerequisite for infection and/or symbiosis tooccur. Consequently, lectin-deficient microbial mutants are often unableto initiate infection. Glycans recognized by microbial surface lectinshave been shown to block the adhesion of bacteria to animal cells invitro and in vivo, and thus may protect animals against infection bybacteria.

In view of the wide range of microorganisms containing various glycanson their cell surface, there is a need to identify natural broadspectrum antimicrobial agents having properties characteristic oflectins for binding and neutralizing microorganisms in microbialinfections in both humans and plants. The present application addressesthis need and provides a plant-derived broad spectrum antimicrobialhevamine A-related protein for use in nutraceutical compositions,methods for preventing or treating microbial infections in both humansand plants, and in the construction of disease-resistant transgenicplants.

SUMMARY

In one aspect, the present application provides a nutraceuticalcomposition comprising an antimicrobial hevamine A-related proteincomprising an amino acid sequence at least 95% identical to SEQ ID NO: 3or SEQ ID NO: 4, and at least one nutraceutically acceptable carrier.

In one embodiment, the nutraceutical composition, further comprising oneor more nutraceutical ingredients selected from the group consisting ofantimicrobial agents, immune-stimulating agents, anti-inflammatoryagent, antioxidant agent, and combinations thereof, wherein the one ormore nutraceutical ingredients comprise zinc and quercetin.

In another embodiment, the nutraceutical composition formulated fororal, intravenous or intramuscular administration.

In another embodiment, the nutraceutical composition is formulated inthe form of a capsule, a tablet or a lozenge.

Another aspect of the present applicant discloses a method of preparingthe nutraceutical composition, comprising the steps of: drying a plantcomprising a protein comprising an amino acid sequence at least 95%identical to SEQ ID NO:4; extracting the dried plant in an aqueousmedium; and separating the aqueous medium from solid material to form anaqueous extract, wherein the aqueous extract comprises the protein.

In one embodiment, the method further comprises the step of purifyingthe protein from the aqueous extract by immunoaffinity purification togenerate a purification product.

In another embodiment, the method further comprising the step of addingone or more nutraceutical ingredients to the purification product,wherein the one or more nutraceutical ingredients are selected from thegroup consisting of antimicrobial agents, immune-stimulating agents,anti-inflammatory agent, antioxidant agent, and combinations thereof,and wherein the one or more nutraceutical ingredients comprise zinc andquercetin.

In another embodiment, the method further comprises the step of: passingthe aqueous extract through a molecular weight cut-off filter;collecting a retentate comprising the protein; and purifying the proteinfrom the retentate to generate a purification product.

In another embodiment, the method further comprises further comprisingthe step of adding one or more nutraceutical ingredients to thepurification product, wherein the one or more nutraceutical ingredientsare selected from the group consisting of antimicrobial agents,immune-stimulating agents, anti-inflammatory agent, antioxidant agent,and combinations thereof, and wherein the one or more nutraceuticalingredients comprise zinc and quercetin.

Another aspect of the present application discloses a method forpreventing or reducing symptoms of a condition in a subject, comprisingadministering to the subject an effective amount of the nutraceuticalcomposition.

In one embodiment, the condition is microbial infection diseases,abnormal high energy metabolism or low energy metabolism.

In another embodiment, the higher energy metabolism is anemia,pregnancy, growth, exercise, cancers, recovery from surgical and otherinjuries.

In another embodiment, the low energy metabolism comprises malnutrition,anorexia, or aging.

In another embodiment, the microbial infection is caused by a virus,wherein the virus is HIV, influenza Type 1 virus, SARS-CoV-2, SARS-CoV-2or MERS-CoV.

In another embodiment, nutraceutical composition is administered to thesubject orally, intravenously or intramuscularly.

Another aspect of the present application discloses a method ofpreventing or treating a microbial plant infection, comprising applyingan effective amount of a composition, either pre- or post-infection, toa plant, plant part, or media in which a plant is growing, wherein thecomposition comprises an antimicrobial hevamine A-related proteincomprising an amino acid sequence at least 95% identical to SEQ ID NO:3or SEQ ID NO:4, wherein the plant part is selected from the groupconsisting of leaves, roots, stems, fruit, seeds, tubers, bulbs,flowers, pods, stems, shoots, and combinations thereof.

In one embodiment, the composition is applied by spraying a liquid orpowder formulation to the plant, plant part, or a medium in which theplant is growing.

The present application further discloses a transgenic plant, transgenicplant part, or transgenic plant cell, comprising: a stably integratedDNA expression construct comprising: a polynucleotide comprising anucleotide sequence at least 95% identical to SEQ ID NO:1 or SEQ IDNO:2, or a polynucleotide encoding a protein comprising an amino acidsequence at least 95% identical to SEQ ID NO:3 or SEQ ID NO:4, whereinthe transgenic plant, transgenic plant part, or transgenic plant cellexhibits increased resistance to at least one bacterial, fungal or viralinfection as compared to a control plant, plant part, or plant celllacking the DNA expression construct.

In one embodiment the transgenic plant, transgenic plant part, ortransgenic plant cell is derived from a crop plant selected from thegroup consisting of wheat, corn, rice, barley, cotton, canola, alfalfa,sugarbeet, potato and tomato.

The present application also describes a method of producing atransgenic plant, comprising the steps of: (a) stably transforming intoa host plant a recombinant DNA expression construct comprising: apolynucleotide comprising a nucleotide sequence at least 95% identicalto SEQ ID NO:1 or SEQ ID NO:2, or a polynucleotide encoding a proteincomprising an amino acid sequence at least 95% identical to SEQ ID NO:3or SEQ ID NO:4; and (b) isolating a transgenic plant expressing thestably transformed polynucleotide in an amount sufficient to provideincreased resistance to a fungal, bacterial or viral infection ascompared to a control plant lacking the polynucleotide under the samecondition, wherein the host plant is a crop plant selected from thegroup consisting of wheat, corn, rice, barley, cotton, canola, alfalfa,sugarbeet, potato and tomato.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary process for producing an aqueous plant extractfrom dried Momordica balsamina leaves.

FIG. 2A shows that water soluble extracts from dried leaves extracts ofMomordica balsamina contain an anti-HIV activity. The water-solubleextracts were tested at concentrations from 0 to 1000 μg/ml and scoredfor infectivity using a MAGI assay. FIG. 2B shows an IC50 determinationof inhibitory activity against HIV-1_(NL43) by exposing 1 ng ofHIV-1_(NL43) to concentrations of MoMo30 from 1 to 100 nM anddetermining the percent infectivity (or inhibition) by MAGI assay. TheIC50 value was determined by curve-fitting using Dr. Fit software. Thetop curve shows MoMo30 inhibition. The bottom curve shows a comparisonto the commercially available HIV inhibitor, Enfuvirtide.

FIGS. 3A and 3B show the effects of different primary solvents (A) orextraction conditions (B) used in processing MoMo30 extracts relative tothe amount of inhibition of HIV as determined by MAGI cell indicatorassays. Lower bars indicate greater inhibition.

FIG. 4A shows the results from using different size molecular weightcutoff filters to either retain (retentate) or pass through (filtrate)the inhibitory product in the plant extract. The plant extract (2 mg/mlstock) was passed through Amicon Ultra cutoff filters (3K, 10K, 30K,100K and control (virus alone) and then mixed with 1 ng of p24equivalent of virus before testing by MAGI assay. In each case theportion that flowed through the filter (solid bars) and a portionretained by the filter (hashed bars) was tested by the MAGI infectivityassay. F=filtrate (passes through filter) and R=retentate (retained byfilter).

FIG. 4B shows that MoMo30 is heat stable and stays bound to virus forlong periods of time. In FIG. 4B, HIV-1NL43 in an amount comprising 1 ngp24 was incubated with MoMo30 at concentrations of 1 nm (bottom curve)and 5 nm (top curve) sufficient to cause 50% or 70% inhibition,respectively. MoMo30 was pre-treated for 30 min at temperatures from 15to 120° C. prior to mixing.

FIG. 4C shows that MoMo30 forms highly stable complexes with HIV-1.MoMo30 (3 nM) was mixed with an amount of HIV-1NL43 comprising 1 ng p24and allowed to interact for 5 min prior to centrifugation through a 40%sucrose cushion. Virus-complexes were removed at times from 5 min to 72h at 4° C. prior to testing for infectivity/blocking by the MAGI cellassay. All measurements were done in triplicate.

FIG. 5A shows that a M. balsamina extract passed through a 30 kD cutofffilter contains a prominent 30 kDa protein as visualized on a Coomasieblue stained 4-20% SDS-PAGE gel. The band is reactive with an N-terminalantibody to MoMo30. FIG. 5B shows that the anti-MoMo30 antibody blockedthe ability of MoMo30 to inhibit HIV-1 infection in a dose-dependentmanner (from 0.5 μg to 5.0 μg).

FIG. 6 shows a Clustal Omega alignment of DNA sequences from M.balsamina MoMo30, the Hevamine A-like protein from M. charantia, and theMAP30 protein from M. charantia. Residues that are different are shaded.MoMo30 is 92% identical to the M. charantia hevamine A-like gene codingregion and 26% identical to the M. charantia MAP30 protein.

FIG. 7A shows the MoMo30 coding region aligned with the hevamineA-related amino acid sequence from Momordica charantia, along withstructural domain predictions thereof. Amino acids highlighted in redshow differences between the two sequences. Arrows denote predicted betasheet structures and hatched boxes denote areas of predicted alphahelical structure. The two yellow shaded boxes denote areas ofconservation in this class of proteins. Asterisks denote highlyconserved catalytic residues. FIG. 7B shows the amino acid sequence ofthe mature MoMo30 (i.e., secreted) protein.

FIG. 8 shows an alignment of two conserved regions from the MoMo30protein against other hevamine A-related proteins.

FIG. 9 , panel A shows a 30 kD in vitro translated MoMo30 product. TheMoMo30 gene was inserted into a pGEM vector that was used as a templatefor coupled transcription/translation. The reaction was run on a 20%SDS-PAGE gel and a western blot was probed with an N-terminal ab toMoMo30. A sample of purified MoMo30 is used as a marker. Panel B showsthat the translation products have anti-HIV activity as determined by aMAGI assay. In panel C, the MoMo30 pGEM plasmid was transfected into HEK293 cells, followed by collection of supernatants and cell lysatestherefrom, which were run on a 20% SDS-PAGE gel and probed with theN-terminal MoMo30 ab. In panel D, 10 μl of cell-free conditioned mediumwas tested for HIV infectivity by the MAGI assay.

FIG. 10 is a Coomasie stained SDS-PAGE showing that the 30 kDa MoMo30protein from extract A (“Ext”) binds to increasing levels of purifiedHIV gp120 (in relative amounts 5, 10, 20, 30 and 40) and induces it toundergo a shift in MW (see “gp120+Ext”). Note the shift in mobility isevident even after boiling in loading buffer and despite the denaturingconditions in the gel.

FIG. 11 is a schematic depiction of a blocking assay to examine whetherMoMo30-containing extracts from M. balsamina inhibit the binding ofpurified HIV gp120 to CD4. Purified fluorescently labeled gp120 (30 μg,ImmunoDx) was added to 1×106 Jurkat T cells either with PBS or a pooledcombination of extracts.

FIG. 12 , panels A-F show the results of a fluorescence binding assay(as depicted in schematic in FIG. 11 ). Briefly, Jurkat T cells aremixed with FITC labeled gp120 either in the absence (panels A-C) orpresence (panels D-F) of extract A. The results show that binding offluorescently labeled gp120 to the surface of Jurkat T-cells (panel A)is inhibited in the presence of the MoMo30-containing plant extracts(panel D). Panels B and E are the same cells stained with DAPI andpanels C and F depict the same cells under phase contrast.

FIG. 13A shows a surface plasmon resonance (SPR) analysis (Biacore)indicating that MoMo30 protein from a cell extract attaches to HIV gp120so as to prevent its interaction with the CD4 receptor. Gp120 wasimmobilized on the gold surface and MoMo30 protein was flowed across thesurface at concentrations from 6 to 200 nM. The assay was done intriplicate on separate days.

FIG. 13B shows that binding of MoMo30 to gp120 is dependent on glycosylresidues on gp120. A Biacore chip was saturated with gp120 and MoMo30(top curves). The gp120-MoMo30 complexes were treated with PNGglycosylase to remove sugar residues from gp120 (bottom curves). Loss ofsugar residues resulted in a decrease in binding.

FIG. 13A shows MoMo30 binding to purified gp120. In FIG. 14A, Gp120 wasbound to a Biacore chip surface and MoMo30 was allowed to flow acrossthe chip at concentrations from 1 nM to 100 nM. Binding was monitored bychanges in surface plasmon resonance. In FIG. 13B, Gp120 pre-treatedwith PNGase F (an N-linked glycosylase) dramatically reduces binding.The three lines represent triplicate measurements.

FIG. 14A shows that mannose blocks the activity of MoMo30. HIV-1NL4-3,400 ng of MoMo30 and different concentrations of D-mannose wereincubated for 5 min at 37° C. and tested by a MAGI cell assay forinhibition of infection. Jurkat cells (1×107) were infected with 300 ngof HIV-1NL4-3 with or without 20 mg of MoMo30.

FIG. 14B shows that exposure of HIV-1 to MoMo30 reduces its reactivitywith the glycan specific mAb 2G12. Virus was harvested at days 5, 8, and12 and concentrated by centrifugation at 125,000×g through a 30% sucrosecushion. The pellet was subjected to SDS-PAGE and an immunoblot was doneusing anti-gp120 glycosyl specific antibody 2G12 and p24 mouse antibody.

FIG. 15 shows inhibition of simian immunodeficiency virus (SIV-mac239)infectivity by MoMo30 cell extracts.

FIG. 16 shows inhibition of Ebola virus (Zaire strain) infectivity inHeLa or HFF cells by MoMo30 cell extracts.

FIG. 17 shows that an HIV-1 pseudotyped with the aMLV envelope proteinis sensitive to MoMo30 inhibition. An env deleted HIV-1 strain (KFS)pseudotyped to contain the MuMLV envelope glycoprotein was tested forinfectivity in the absence (left) or presence (right) of MoMo30.

FIG. 18 shows adsorption of MoMo30 to the serum of Rhesus macaques. Twomacaques were given herbal therapy in the same regimen as that given inthe field (adjusted for weight). Three microliters of serum was testedby the MAGI assay (in triplicate) for antiviral effects at times from 0to 183 days. The inset shows a western blot using N-terminal MoMo30 aband 15 μl the sample in crosshatched bars.

FIG. 19 shows the results of an MTT assay demonstrating a lack ofcellular toxicity by MoMo30 protein at concentrations between 1 to 1000nm.

FIG. 20A shows that MoMo30 causes hemagglutination. Purified MoMo30 wastested for its ability to agglutinate sheep red blood cells (RBCs). Asshown in panel A, the stock solution at a dilution of 1:512 was found tocause hemagglutination. FIG. 20B shows that MoMo30 stimulates T cellgrowth. In each experiment, a fixed number of Jurkat cells was treated(left to right) with either PBS (control, Con), phytohemagglutinin A(PHA) or an equal amount of MoMo30.

FIGS. 21A-21B show the results of a clinical study (n=61) in which HIV-1infected patients were orally administered an herbal tea daily for 6months containing Extracts A-E above. The results of this study showed adecrease in patients' HIV viral loads following a 6-month treatment withMoMo30 plant extract (FIG. 21A). FIG. 21B shows an increase in CD4+lymphocytes following treatment with the MoMo30 plant extract.

FIG. 22 , panels A and B further show the results of the clinical studydepicted in FIGS. 21A-21B where an increase in CD4+ lymphocytes of about50% was observed following 6 months of treatment with the (FIG. 22 ,panel A), and a decrease of 60% of the patients' mean HIV viral loadswas observed following a 6-months post-treatment (FIG. 22 , panel B),which typically decreased to undetectable levels after 180 months (FIG.22 , panel B). In FIG. 22 , panel C, a subset of the originally treatedpatients (n=13) were re-tested at 180 months. The results of thisanalysis showed that CD4 counts in most of the re-tested patientsreturned to near baseline levels. In addition, viral loads in ten ofthese re-tested patients had decreased to undetectable (<20 copies/ml);two patients had very low levels (˜3000 copies/ml) and one was reportedas (20 copies/ml) at 180 months post-treatment.

FIG. 23 , panels A and B show that the 13 re-tested patients in FIG. 22, panel C produced neutralizing antibodies. FIG. 23 , panels A and Bshow the results of patients' serum being tested for neutralizingactivity against HIV-1 pseudotyped with an HIV-1NL4-3 env or an aMLVenv, respectively. FIG. 23 , panel C shows a table depicting examples ofantibody titers against 10 primary strains and 3 lab strains of HIV-1.The table summarizes reciprocal dilutions of the inhibitory dose toinduce 50% reduction in replication of virus (ID 50). Darker shadedareas depict higher titers, while the lighter shaded areas depict lowertiters.

FIG. 24 shows the results of an analysis in which serum from twopatients (PROM050 and PROM052) treated with MoMo30 extracts were testedfor neutralizing activity against HIVNL4-3 following 3 successive roundsof Protein A/G adsorption. Following adsorption, neutralizing activitywas completely depleted.

While the present disclosure will now be described in detail, and it isdone so in connection with the illustrative embodiments, it is notlimited by the particular embodiments illustrated in the figures and theappended claims.

DETAILED DESCRIPTION OF THE INVENTION

The invention and accompanying drawings will now be discussed inreference to the numerals provided therein to enable one skilled in theart to practice the present invention. The skilled artisan willunderstand, however, that the inventions described below can bepracticed without employing these specific details, or that they can beused for purposes other than those described herein. Indeed, they can bemodified and can be used in conjunction with products and techniquesknown to those of skill in the art considering the present disclosure.The drawings and descriptions are intended to be exemplary of variousaspects of the invention and are not intended to narrow the scope of theappended claims. Furthermore, it will be appreciated that the drawingsmay show aspects of the invention in isolation and the elements in onefigure may be used in conjunction with elements shown in other figures.

It will be appreciated that reference throughout this specification toaspects, features, advantages, or similar language does not imply thatall the aspects and advantages may be realized with the presentinvention should be or are in any single embodiment of the invention.Rather, language referring to the aspects and advantages is understoodto mean that a specific aspect, feature, advantage, or characteristicdescribed in connection with an embodiment is included in at least oneembodiment of the present invention. Thus, discussion of the aspects andadvantages, and similar language, throughout this specification may, butdo not necessarily, refer to the same embodiment.

The described aspects, features, advantages, and characteristics of thepresent application may be combined in any suitable manner in one ormore further embodiments. Furthermore, one skilled in the relevant artwill recognize that the invention may be practiced without one or moreof the specific aspects or advantages of a particular embodiment. Inother instances, additional aspects, features, and advantages may berecognized and claimed in certain embodiments that may not be present inall embodiments of the invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this application belongs. One of skill in the art willrecognize many techniques and materials similar or equivalent to thosedescribed here, which could be used in the practice of the aspects andembodiments of the present application. The described aspects andembodiments of the application are not limited to the methods andmaterials described.

Definitions

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contentclearly dictates otherwise.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to “the value,” greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed.

As used herein, the terms “hevamine A-related protein” and “MoMo30protein” are used interchangeably with reference to an antimicrobialprotein that can be isolated from e.g., Momordica balsamina leaves. Inpreferred embodiments, this protein comprises an amino acid sequence atleast 95% identical to SEQ ID NO: 3 or SEQ ID NO: 4. In certainembodiments, the MoMo30 protein is obtained from a plant of theMomordica genus or a species therefrom, such as Momordica balsamina andothers described herein, or any plant comprising a homolog thereof.

The term “hevamine A-related composition” refers to a compositioncomprising a hevamine A-related protein or MoMo30 protein.

As used herein, the term “MoMo30 homolog” refers to a MoMo30-relatedprotein that is at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to the amino acid sequence of the Momordicabalsamina MoMo30 protein set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

The phrase “antimicrobial agent”, “antimicrobial product” or“antimicrobial protein” are used interchangeably with reference toprotein or small molecule compound that can inhibit the progression of amicrobial infection, including those caused by viruses, bacteria, andfungi, or induce or mediate the death (e.g., necrosis or apoptosis) ofinfected cells in a subject (e.g., a human).

As used herein, the “subject is mammal or human.”

As used herein, the term “nutraceutical composition” refers to acomposition containing hevamine A-related protein or MoMo30 protein,optionally with one or more nutraceutical ingredients. The phrases“nutraceutical composition comprises” and “nutraceutical compositioncomprising” should be interpreted such that the “comprises” or“comprising” components are included in a single nutraceuticalcomposition or in one or more independent nutraceutical compositions.

As used herein, the term “nutraceutical ingredient” is used withreference to any natural compound, substance, extract, or food that actsas a pharmaceutical agent alternative exhibiting at least one medical orhealth benefit when administered to a subject. Preferably, the medicalor health benefit corresponds to an antimicrobial and/orimmune-stimulating property. As used herein, the nutritional ingredientis added to a nutraceutical composition containing MoMo30. Exemplarynutraceutical ingredients include but are not limited to, antimicrobialagents, immune-stimulating agents, anti-inflammatory agent, antioxidantagent, and combinations thereof. The natural ingredients thereof may beprepared from natural sources, or they may be synthetically synthesized.The nutraceutical ingredients and MoMo30 protein can be contained in amedicinal format such as a capsule, tablet, or powder in a prescribeddose, or in a liquid or beverage.

As used herein, the phrase “nutraceutically acceptable carrier,” refersto a substance that is not biologically or otherwise undesirable, i.e.,the substance may be incorporated into a nutraceutical compositionadministered to a patient without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the composition in which it is contained. Further, thenutraceutically acceptable carrier is used with reference to a nontoxic,inert solid, semi-solid, diluent, encapsulating material or formulationauxiliary of any type.

As used herein, the term “antioxidant” refers to natural substance thatprevents or delays the oxidative deterioration of a compound.

The terms “treat” and “treatment” refer to the amelioration of one ormore symptoms associated with a coronavirus infection; prevention ordelay of the onset of one or more symptoms of a viral infection; and/orlessening of the severity or frequency of one or more symptoms of theinfection.

The term “effective amount” is used with reference to the amount(s) ofone or nutraceutical ingredients needed to provide a threshold level ofactive ingredients in the bloodstream or target tissue to provide aprophylactic or therapeutic effect. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. The precise amount of nutraceutical ingredients willdepend upon numerous factors, e.g., the particular active agent, thecomponents and physical characteristics of the composition, intendedpatient population, patient considerations, and the like, and canreadily be determined by one skilled in the art, including based uponthe information provided herein or otherwise available in the relevantliterature.

The terms “codon optimized” and “codon optimization” refer to a processfor modifying a nucleic acid sequence according to one or more of thefollowing: (1) to match codon frequencies in a host organism target; (2)to promote increased expression; (3) to ensure proper folding; (4) toprovide a GC content suitable for increasing mRNA stability or reducingsecondary structures; (5) to minimize tandem repeat codons or base runsthat may impair gene construction or expression; (6) to customizetranscriptional and translational control regions; (7) to insert orremove protein trafficking sequences; (8) to remove/add post translationmodification sites in an encoded protein (e.g. glycosylation sites); (9)to add, remove or shuffle protein domains; (10) to insert or deleterestriction sites; (11) modify ribosome binding sites and mRNAdegradation sites; (12) to adjust translational rates to allow thevarious domains of the protein to fold properly; or (13) to reduce oreliminate problem secondary structures within the polynucleotide. Codonoptimization tools, algorithms and services are known in theart—non-limiting examples include services from GeneArt (LifeTechnologies), DNA2.0 (Menlo Park Calif.) and/or proprietary methods.

The terms, “improve”, “increase” or “reduce”, as used in this context,indicate values or parameters relative to a baseline measurement, suchas a measurement in the same individual prior to initiation of thetreatment described herein, or a measurement in a control individual (ormultiple control individuals) in the absence of the treatment describedherein.

The term “control individual” is an individual who is not afflicted withthe same microbial infection as the individual being treated, who isabout the same age as the individual being treated (to ensure that thestages of the disease in the treated individual and the controlindividual(s) are comparable). The individual (also referred to as“patient” or “subject”) being treated may be a fetus, infant, child,adolescent, or adult human.

Further, it should be understood that any reference to “HIV” or “HIV-1”should be construed as applying to any isolate or clade of HIV-1 orHIV-2.

I. Nutraceutical Compositions and Methods

The present application is directed to a nutraceutical compositioncomprising an antimicrobial hevamine A-related protein from plants,which has been found to possess antiviral, antibacterial and antifungalproperties. The hevamine A-related protein may be orally administeredalone, or preferably in combination with one or more nutraceuticalingredients further descried below.

In one aspect, the present application relates to a nutraceuticalcomposition comprising an antimicrobial hevamine A-related proteincomprising an amino acid sequence at least 90%, at least 95% identical,at least 99%, or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4, and atleast one nutraceutically acceptable carrier for oral administration.

The MoMo30 product from Momordica balsamina is characterized by multipleproperties, including: (1) an amino acid sequence of SEQ ID NO: 3 or SEQID NO: 4; (2) a size of about 30 kDa; (3) soluble in aqueous solutions;(4) high heat resistance or high stability as reflected in noappreciable loss of activity following autoclaving at 120° C. for 30min; (5) mannose-sensitive binding to HIV gp120; (6) insensitive todigestion with trypsin following denaturation in 8M urea and overnighttreatment and partially sensitive to subtilisin after overnighttreatment; (7) an IC50 of about 32 pM in a MAGI cell indicator assay;(8) hemagglutinin activity; (9) capable of activating and stimulating Tcell proliferation; (10) having chitinase activity; and (11) capable ofpreventing infection by HIV-1 or alleviating symptoms in an HIV-1infected patient.

Without wishing to be bound by theory, MoMo30 is believed to be acarbohydrate binding agent with two distinct modes of action: (1)inhibition of virus by blocking entry into cells; (2) selecting formutations in the viral envelope that allow the host to produce a broadlyneutralizing antibody response. MoMo30 inhibits virus through bindingcarbohydrates. The more carbohydrates on the gp120, the more targetswill be available for inhibiting virus. Under such pressure, thepresence of the MoMo30 selects for virus with fewer glycosyl groups.Fewer glycosyl groups on gp120 allow more epitopes to be exposed andallows the production of neutralizing antibodies. Consequently, patientstreated with MoMo30 in the short-term exhibit the production of abroadly neutralizing antibody response. The same patients should alsodevelop a broadly neutralizing antibody response to control theirinfection in the long term.

In some embodiments, the MoMo30 protein (or homolog thereof) is encodedby a plant species of the Momordica genus. Exemplary Momordica speciesinclude, but are not limited to, M. aculeata, M. acuminate, M.acutangula, M. adoensis, M. affinis, M. amaniana, M. angolensis, M.angulate, M. angustisepala, M. anigosantha, M. anthelmintica, M.argillicola, M. aspera, M. auriculata, M. balsamina, M. bequaertii, M.bicolor, M. boivinii, M. brachybotrys, M. bracteata, M. brevispinosa, M.bricchettii, M. cabraei, M. calantha, M. calcarata, M. camerounensis, M.cardiospermoides, M. carinata, M. casea, M. charantia, M. chinensis, M.cirrhiflora, M. cissoides, M. clarkeana, M. clematidea, M.cochinchinensis, M. cochinchinensis, M. cogniauxiana, M. cordata, M.cordatifolia, M. coriacea, M. corymbifera, M. covel, M. crinocarpa, M.cucullata, M. cylindrica, M. cymbalaria, M. dasycarpa, M. denticulata,M. denudata, M. dictyosperma, M. dioica, M. diplotrimera, M. dissecta,M. eberhardtii, M. echinata, M. echinocarpa, M. ecirrhata, M. elastica,M. elaterium, M. elegans, M. enneaphylla, M. erinocarpa, M. fasciculata,M. foetida, M. friesiorum, M. gabonii, M. garipensis, M. garriepensis,M. gilgiana, M. glabra, M. glauca, M. gracilis, M. grandibracteata, M.grosvenorii, M. guttata, M. hamiltoniana, M. hamiltoniana, M.henriquesii, M. heterophylla, M. heyneana, M. hispida, M. huberi, M.humilis, M. hystrix, M. indica, M. involucrata, M. jagorana, M.jeffreyana, M. kirkii, M. lambertiana, M. lanata, M. laotica, M.laurentii, M. leiocarpa, M. littorea, M. luffa, M. luffa, M. macrantha,M. macropetala, M. macrophylla, M. macropoda, M. macrosperma, M.maculata, M. mannii, M. marlothii, M. martinicensis, M. meloniflora, M.microphylla, M. missionis, M. mixta, M. monadelpha, M. morkorra, M.mossambica, M. multicrenulata, M. multiflora, M. muricata, M.obtusisepala, M. officinarum, M. operculata, M. ovata, M. paina, M.palmata E, M. papillosa, M. parvifolia, M. pauciflora, M. pedata, M.pedisecta, M. peteri, M. procera, M. pterocarpa, M. punctata, M.purgans, M. pycnantha, M. quinquefida, M. quinqueloba, M. racemiflora,M. racemosa, M. renigera, M. repens, M. reticulata, M. rostrata, M.rotunda, M. roxburghiana, M. rumphii, M. runssorica, M. rutshuruensis,M. sahyadrica, M. sativa, M. schimperiana, M. schinzii, M. schliebenii,M. senegalensis, M. sessilifolia, M. sicyoides, M. silvatica, M.sinensis, M. somalensis, M. sphaeroidea, M. spicata, M. spinosa, M.stefaninii, M. subangulata, M. surculata, M. suringarii, M. thollonii,M. tonkinensis, M. trifolia, M. trifoliata, M. trilobata, M. tuberosa,M. tubiflora, M. tubulosa, M. umbellata, M. verticillata, M. vogelii, M.wallichii, M. welwitschii, M. wildemaniana, M. zeylanica, and M.zeylanica. In some embodiments, the MoMo30 protein may be obtained fromany of the foregoing Momordica leaf extracts, fruit extracts, rootextracts, bark extracts, seed extracts and/or any flower thereof.

In preferred embodiments, the nutraceutical composition is obtained fromMomordica balsamina leaf extracts. In other embodiments, thenutraceutical composition is obtained from Momordica balsamina fruitextracts, root extracts, bark extracts, seed extracts and/or any flowerthereof.

In one embodiment, the nutraceutical composition contains a MoMo30protein or MoMo30 homolog comprising an amino acid sequence that is atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO:4 and contains at least one amino acid substitution relative SEQ ID NO:3or SEQ ID NO: 4, respectively.

In some embodiments, the MoMo30 protein is a variant containing one ormore mutations relative to the wild-type sequence. “Variants” includeprotein sequences having one or more amino acid additions, deletions,stop positions, or substitutions, as compared to a wild-type protein. Anamino acid substitution can be a conservative or a non-conservativesubstitution. Variants of MoMo30 proteins can include those having oneor more conservative amino acid substitutions. A “conservativesubstitution” or “conservative amino acid substitution” involves asubstitution found in one of the following conservative substitutionsgroups: Group 1: Alanine (Ala; A), Glycine (Gly; G), Serine (Ser; S),Threonine (Thr; T); Group 2: Aspartic acid (Asp; D), Glutamic acid (Glu;E); Group 3: Asparagine (Asn; N), Glutamine (Gln; Q); Group 4: Arginine(Arg; R), Lysine (Lys; K), Histidine (His; H); Group 5: Isoleucine (Ile;I), Leucine (Leu; L), Methionine (Met; M), Valine (Val; V); and Group 6:Phenylalanine (Phe; F), Tyrosine (Tyr; Y), Tryptophan (Trp; W).

Additionally, amino acids can be grouped into conservative substitutiongroups by similar function, chemical structure, or composition (e.g.,acidic, basic, aliphatic, aromatic, or sulfur-containing). For example,an aliphatic grouping may include, for purposes of substitution, G, A,V, L, and I. Other groups including amino acids that are consideredconservative substitutions for one another include: sulfur-containing: Mand C; acidic: D, E, N, and Q; small aliphatic, nonpolar or slightlypolar residues: A, S, T, P, and G; polar, negatively charged residuesand their amides: D, N, E, and Q; polar, positively charged residues: H,R, and K; large aliphatic, nonpolar residues: M, L, I, V, and C; andlarge aromatic residues: F, Y, and W.

Non-conservative substitutions include those that affect the structureof the peptide backbone in the area of alteration (e.g., thealpha-helical or beta-sheet structure); the charge or hydrophobicity ofthe molecule at the target site; or the bulk of the side chain.Non-conservative substitutions which in general are expected to producethe greatest changes in a protein's properties may include those inwhich e.g., (i) a hydrophilic residue (e.g., S or T) is substituted for(or by) a hydrophobic residue (e.g. L, I, F, V, or A); (ii) a C or P issubstituted for (or by) any other residue; (iii) a residue having anelectropositive side chain (e.g. K, R, or H) is substituted for (or by)an electronegative residue (e.g., Q or D); or (iv) a residue having abulky side chain (e.g., F) that is substituted for (or by) one nothaving a bulky side chain, (e.g., G).

MoMo30 mutants may be generated by random mutagenesis or site-directedmutagenesis using methods known to those of ordinary skill in the artwith or without selection methodologies employing MAGI indicator cellassays, apoptosis assays and the like.

In one embodiment, the MoMo30 protein or MoMo30 homolog comprises anamino acid sequence that is at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to the amino acid sequence ofSEQ ID NO: 3 or SEQ ID NO: 4 and contains at least one amino acidsubstitution relative SEQ ID NO:3 or SEQ ID NO: 4, respectively.

In another aspect, the present application provides an expression vectorcomprising a MoMo30-encoded nucleic acid or a codon-optimized nucleicacid for expressing a MoMo30 protein. In certain embodiments, theexpression vector encodes a MoMo30 protein containing at least one aminoacid substitution relative SEQ ID NO:3 or SEQ ID NO: 4.

The MoMo30 protein may be derived from bacterial, fungal, plant, insect,or animal cells transformed with a MoMo30 expression vector to expressthe protein. The transformed cells may be stably transformed, or theymay be transiently transformed. The MoMo30 protein or extract may beprepared, and its composition may be modified in accordance with any ofthe methods of preparation outlined below or known to those of ordinaryskill in the art.

In some embodiments, the bacterial, fungal, plant, insect, or animalcells are transformed with a MoMo30 expression vector containing aMoMo30 encoded nucleic acid that is at least 95% identical to thenucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In someembodiments, the nucleotide sequence comprises at least nucleotidesubstitution relative the nucleotide sequence of SEQ ID NO: 1 or SEQ IDNO: 2. In some embodiments, the MoMo30-encoded nucleic acid includes acodon-optimized nucleic acid coding region.

In some embodiments, the MoMo30 protein of the present application isexpressed from a MoMo30 expression vector containing a codon-optimizednucleic acid coding region. In certain embodiments, the nucleic acid iscodon optimized for expression in plant cells. In certain embodiments,the nucleic acid is codon optimized for expression in mammalian or humancells. In certain embodiments, the nucleic acid is codon optimized forexpression in insect cells. In certain embodiments, the nucleic acid iscodon optimized for expression in bacteria. In certain embodiments, thenucleic acid is codon optimized for expression in fungal cells.

Codon optimization methods are known in the art and may be used asprovided herein. In some embodiments, the open reading frame (ORF)sequence in a polynucleotide is optimized using optimization algorithmsas described herein and known in the art.

In some embodiments, the codon optimized MoMo30 polynucleotide sequenceshares less than 95%, less than 90%, less than 85%, less than 80%, lessthan 75%, less than 70%, less than 65%, less than 60%, less than 55% orless than 50% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

In some embodiments, the codon optimized polynucleotide sequence sharesbetween 50% and 95%, between 50% and 90%, between 50% and 85%, between50% and 80%, between 50% and 75%, between 50% and 70%, between 50% and65%, between 50% and 60%, between 50% and 55%, between 55% and 95%,between 55% and 90%, between 55% and 85%, between 55% and 80%, between55% and 75%, between 55% and 70%, between 55% and 65%, between 55% and60%, between 60% and 95%, between 60% and 90%, between 60% and 85%,between 60% and 80%, between 60% and 75%, between 60% and 70%, between60% and 65%, between 65% and 95%, between 65% and 90%, between 65% and85%, between 65% and 80%, between 65% and 75%, between 65% and 70%,between 70% and 95%, between 70% and 90%, between 70% and 85%, between70% and 80%, between 70% and 75%, between 75% and 95%, between 75% and90%, between 75% and 85%, between 75% and 80%, between 80% and 95%,between 80% and 90%, between 80% and 85%, between 85% and 95%, between85% and 90%, or between 90% and 95% sequence identity to SEQ ID NO: 1 orSEQ ID NO: 2.

Nutraceutical Ingredients

In certain embodiments, the nutraceutical composition includes a MoMo30protein or MoMo30-containing extract that is combined with one or moreadditional nutraceutical ingredients, particularly those exhibitingantimicrobial, antiviral, antibacterial, antifungal, immune boosting,anti-inflammatory, and/or antioxidant properties. Such nutraceuticalsmay include one or more phytochemicals, minerals and metals, vitamins,salts, amino acids, fatty acids, proteins, and other nutraceuticallyacceptable excipients.

Plants provide a prominent source of bioactive phytochemicals.Phytochemicals are produced during the natural course of plant growthand development that provide protection against microorganisms, insectsor herbivores and serve as plant defense mechanisms againstenvironmental stressful conditions, and. Phytochemicals such ascarotenoids, phenolics, alkaloids, and organosulfur compounds arecurrently marketed for various medical or health benefits.Phytonutrients from many indigenous plants have been evaluated forantimicrobial, immune stimulating, anticancer, cardioprotective andbrain enhancing effects. Many plants contain several distinctphytochemicals which may interact with multiple biological targets andprovide numerous medical and health benefits for humans.

Phytochemicals extracted from plants or herbs include e.g., flavonoids,flavonols, terpenoids, lignans, sulfides, phenols, polyphenols,coumarins, saponins, furyl compounds, alkaloids, thiophenes, essentialoils, alkaloids, lectins, proteins and peptides, and are present involatile essential oils plants, herbs, spices, and herbal teas.

In some embodiments, the nutraceutical composition includes one or moreherbs or herbal ingredients selected from the group consisting oforegano (including oregano oil and carvacrol), sage (includingsafficinolide and sage one), basil (including apigenin and ursolicacid), fennel (including trans-anethole), garlic, goldenseal, lemonbalm, peppermint (including menthol and rosmarinic acid), rosemary(including oleanolic acid), echinacea, sambucus, black cumin seed, teatree oil, olive leaf, myrrh extract, turmeric, licorice, astragalus,ginger, ginseng, dandelion, berberine, and combinations thereof.Preferably the herbs and herbal ingredients have antimicrobial and/orimmune-boosting properties.

In certain embodiments, the present application relates to the methodsin treating the conditions by administering a subject in need aneffective amount of the nutraceutical composition.

Conditions included but not limited to, i.e., microbial infection,abnormal high energy metabolism, i.e., anemia, pregnancy, growth,exercise, cancers; infectious disease and recovery from surgical andother injuries; low energy metabolism, i.e., malnutrition, anorexia, andaging.

In some embodiments, the nutraceutical composition includes one or moreplant extracts or plant substances therefrom. Exemplary plant extractsor sources of MoMo30 homologs may be obtained from one or more membersselected from the group consisting of Acacia arabia, Afromomummelegueta, Agrimonia eupatoria, Ajuga decumbens, Allium cepa, Alliumsativum, Aloe vera, Alternanthera philoxeroides or sessiles, Ammi maius,Andographis paniculata, Apium graveolens, Apium leptophyllum, Arachishypogaea, Arctium lappa, Artemesia Judaica, Amebia euhcroma, Asparagusracemosus, Astragalus spinosus, Astragalus lentingosis swainsonine,Azadirachta indica, Balanites aegyptiaca, Bauhinia rufescens, Bersamatysoniana, Blumea alata, Brucea antidysenterica, Buchenavia capita,Butyrospermum parkii, Bryonia cretica ssp. Dioica, Bryonia angustifolia,Calotropis procera, Camellia sinensis (green tea extract), Camelliatheifera, Casia sieberiana, Catha edulis. Cedrela toona, Chrysanthemummorifolium, Cinnamomum verum, Citrus limonia, Clausena anisata, Cliviaminiata, Cochlospermum planchonii, Coffea arabica, Cola nitida,Combretum glutinosum, Combretum micranthum, Coptis chinesis, Coptisteetoides, Coptis japonica, Coraria nepalensis, Coriandrum sativum,Cryptolepis sanguinolenta, Curcuma longa, Cyperus articulatus, Cyperusdomestus, Cyperus rigidifolius, Datura metel syn alba, Daucus carota,Diospyros mespiliformis, Echinacea angustiflora, Echinacea purpurea,Echinacea simulata, Echinacea pallida, Elettaria cardamomum, Entadaabyssinica, Epimedium grandiflorum, Epimedium sagittatum, Epimediumsinense, Epilobium angustifolium, Erigeron Canadensis, Eugenia orSyzigium claviflorum, Euphorbia hirta, Faidherbia albida, Fagaraxanthox, Ficus iteophylla, Ficus platphylla, Foeniculum vulgarel,Garcinia afzelii, Garcinia epundata, Gardenia coronaria, Gaultheriatrichophylla, Glycine max, Glycyrrhiza glabra, Gossypium herbaceum,Guiera senegalensis, Heracleum sphondylium, Hypericum perforatum,Hypericum japonicum, Hyssopus officinalis, Jasminum officinale, Khayasenegalensis, Lippia javanica, Lithospermum erythrorhizon, Lonicerajaponica, Lophira lanceolate, Luffa, Lycopus europaeus, Magnoliaofficinalis, Mallotus repandus, Mallotus philippinesis, Matricariachamomil, Matricaria recutitia, Melissa parviflora, Melissa officinalis,Momordica species, including Momordica balsamina, Momordica charantiaand others; Morus nigra (black mulberry), Morus rubra, Morinda lucida,Narcissus tazetta, Narcissus pseudonarcissus, Nigella sativa, Ocimumtenuiflorum, Ocimum gratissimum, Oenthera rosea, Paeonia spec., Panaxginseng, Papaver somniferum, Parkia biglobosa, Perilla frutescens,Persea americana, Phyllanthus amarus, Phyllanthus emblica Phyllanthusniruri, Pimpinella anisum, Pinus koraicenis, Pinus maritima, Pinusparviflora, Piper nigrum, Plumeria rubra, Polyantha suberosa, Prosopissp., including P. africana and others; Prunus africans, Prunellavulgaris, Prunus bakariensis, Prunus amygdalus, Psoralea corylifolia,Randia dunatorum, Raphanus sativus, Rheum palmatum, Rhus coriaria, Rhuschinesis, Ricinus communis, Rosmarinus officinalis, Salic mucronata,Salvia miltiorhiza, Salvia officinalis, Salvadora persica, Sambucuscanadensis, Sambucus ebulus, Sambucus nigra (elderberry), Saussurealappa, Scilla griffrthii, Scutellaria baicalensis baiealein, Sedumsediforme, Senecio scandens, Senecio aereus, Senna alata, Silybummarianum, Skimmia laureola, Solarium niporum, Stevia rebaudiana, Swertiafranchetiana, Syzygium aromaticum, Syzygium cumini, Tamarindus indica,Terminalia alata Terminalia catappa, Terminalia chebula, Terminaliaglaucescens, Thula occidentalis, Tinospora cordifoila, Trapalaponicaspec., Trichosanthes dioica, Trichosanthes kirilowii, Uncaria tomentosa,Urtica dioica, Viola yeodensis, Vitellaria paradoxa, Voacanga africana,Withania somnifera, Woodfordia fruticosa, Woodwardia spec., Zanoxylumnitidum, Zanthoxylum zanthoxyloides, Zingiber officinale, and Ziziphusmauritania, including extracts and polyphenols therefrom. Plant extractsand polyphenols therefrom may be included in powders and liquids of thepresent application, and may be extracted from leaves, bark, seeds,roots, fruits and/or flowers of plants. In some instances, thepolyphenols and other natural nutraceutical ingredients described hereinmay be synthetically produced.

Exemplary plant-derived substances for inclusion in the nutraceuticalcompositions of the present application include lentinan, apolysaccharide isolated from the fruit body of shiitake mushroom(Lentinula edodes mycelium) and various ribosome inactivating proteins(RIPs) from e.g., M. balsamina and Trichosanthis kirilowii, such asMomordin I and Momordin II, as well as ribosome inactivating proteins(RIPs) from any of the foregoing plant extracts. It is believed that theaddition of the nutraceutical ingredients may further increase theprophylactic and/or therapeutic efficacy of the MoMo30 protein,especially in patients with microbial infections or susceptible tomicrobial infections, such as the microbial infections described herein.

In some embodiments, the nutraceutical composition includes one or morenatural substances with antimicrobial and/or immune-enhancing activityselected from the group consisting of quercetin, kaempferol, curcumin,hesperidin, resveratrol, lactoferrin, cinnamaldehyde, allicin, piperine,phycocyanobilin, astaxanthin, propolis, acetyl-L-carnitine, luteolin,apigenin, glycyrrhizin, caffeic acid, chlorogenic acid, epigallocatechingallate (EGCG), Coenzyme Q10, omega-3 fatty acids, N-acetyl cysteine,colloidal silver, baicalin, linalool, eugenol, β-caryiphyllene,p-cymene, gingerol, allylisothiocyanate, carvacrol, o-cymophenol,capsaicin, carnosic acid, carnosol, rosmarinic acid, cineol, eucalyptol,thymol, nisin, β,βdimethylacryl shikonin and acetyl shikonin fromAchillea nobilis; ursolic acid from e.g., rosemary and thyme; vasicinefrom e.g., Adhatoda vasica; sitosterol-D-glucopyranoside from e.g.,Desmostachya bipinnata; probiotics (e.g., Lactobacillus sp., such as L.paracasei, L. plantarum, L. rhamnosus, L. acidophilus; Lactococcuslactis, L. reuteri, B. bifidum, and L. bulgaricus; Bifidobacteriumbifidum and Streptococcus thermopholis).

In some embodiments, the nutraceutical composition includes one or moreimmune-stimulating agents selected from vitamin C, vitamin D, green teaextract, zinc, quercetin, elderberry, N-acetyl cysteine, baicalin,ferulic acid, lipoic acid, Brewer's yeast beta-glucan, and glucosamine.

In some embodiments, the nutraceutical composition includes one or moremicronutrients or minerals, such as zinc, zinc citrate, zinc oxide,selenium, calcium, manganese gluconate, copper, copper gluconate,folate, iron, magnesium, boron sulfate, glucosamine sulfate, andchromium polynicotinate, including salts thereof.

In some embodiments, the nutraceutical composition includes one or morevitamins. Exemplary vitamins include, but are not limited to vitamin A,vitamin B 1, vitamin B2, vitamin B5, vitamin B6, vitamin B7 (biotin),vitamin B9 (folate), vitamin B12, vitamin C, vitamin D, vitamin E, andvitamin K.

In some embodiments, the nutraceutical composition includes one or morefatty acids selected from the group consisting of linoleic acid (LA),gamma linoleic acid (GLA), eicosapentaneoic acid (EPA), docosapentaneoicAcid (DPA), docosahexaenoic acid (DHA), palmitoylethanolamide,short-chain fatty acids (SCFA), medium-chain fatty acids (MCFA), andD-alpha-tocopherol.

In some embodiments, the nutraceutical composition includes one or moreantioxidants. As used herein “antioxidant” refers to any substance whichinhibits, suppresses, or reduces oxidative damage to cells andbiomolecules. Examples of suitable antioxidants for embodiments of thisapplication include, but are not limited to, vitamins, vitamincofactors, minerals, hormones, carotenoids, carotenoid terpenoids,non-carotenoid terpenoids, flavonoids, flavonoid polyphenolics (e.g.,bioflavonoids), flavonols, flavones, phenols, polyphenols, esters ofphenols, esters of polyphenols, nonflavonoid phenolics, isothiocyanates,vitamin A, vitamin C, vitamin E, ubiquinone, mineral selenium,manganese, melatonin, α-carotene, β-carotene, lycopene, lutein,zeanthin, crypoxanthin, reservatol, eugenol, quercetin, glutathione,catechin, gossypol, hesperetin, curcumin, ferulic acid, thymol,hydroxytyrosol, spirulina, turmeric, thyme, olive oil, lipoic acid,glutathione, glutamine, oxalic acid, tocopherol-derived compounds,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),ethylenediaminetetraacetic acid (EDTA), tert-butylhydroquinone, aceticacid, pectin, tocotrienol, tocopherol, coenzyme Q10, zeaxanthin,astaxanthin, canthaxantin, saponins, limonoids, kaempfedrol, myricetin,isorhamnetin, proanthocyanidins, quercetin, rutin, luteolin, apigenin,tangeritin, hesperetin, naringenin, erodictyol, flavan-3-ols (e.g.,anthocyanidins), gallocatechins, epicatechin and its gallate forms,epigallocatechin and its gallate forms (ECGC) theaflavin and its gallateforms, thearubigins, isoflavone, phytoestrogens, genistein, daidzein,glycitein, anythocyanins, cyanidin, delphinidin, malvidin, pelargonidin,peonidin, petunidin, ellagic acid, gallic acid, salicylic acid,rosmarinic acid, cinnamic acid and its derivatives (e.g., ferulic acid),chlorogenic acid, chicoric acid, gallotannins, ellagitannins,anthoxanthins, betacyanins and other plant pigments, silymarin, citricacid, lignan, antinutrients, bilirubin, uric acid, R-a-lipoic acid,N-acetyl cysteine, emblicanin, apple extract, apple skin extract(applephenon), rooibos extract red, rooibos extract, green, hawthornberry extract, red raspberry extract, green coffee antioxidant (GCA),aronia extract 20%, grape seed extract (VinOseed), cocoa extract, hopsextract, mangosteen extract, mangosteen hull extract, cranberry extract,pomegranate extract, pomegranate hull extract, pomegranate seed extract,hawthorn berry extract, pomella pomegranate extract, cinnamon barkextract, grape skin extract, bilberry extract, pine bark extract,pycnogenol, elderberry extract, mulberry root extract, wolfberry (gogi)extract, blackberry extract, blueberry extract, blueberry leaf extract,raspberry extract, turmeric extract, citrus bioflavonoids, blackcurrant, ginger, acai powder, green coffee bean extract, green teaextract, and phytic acid, or combinations thereof.

In some embodiments, the nutraceutical composition is in dried form. Inparticular embodiments, the dried form is a powdered form, granulatedform, capsule, tablet, lozenge, or herbal tea extract.

In some embodiments, the present application provides a liquidcontaining a dried form of the nutraceutical composition in beverage,such as water, carbonated water, juice, herbal tea, soft drinks, energydrinks, or milk. In certain embodiments, the beverage provides one ormore natural sweeteners.

Liquid preparations for oral administration can take the form of, forexample, elixirs, solutions, syrups or suspensions, or they can bepresented as a dry product for constitution with water or other suitableliquids described herein before use. Such liquid preparations can beprepared by conventional means with nutraceutically acceptable additivessuch as suspending agents (e.g., sorbitol syrup, cellulose derivativesor hydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol, or fractionated vegetable oils); and preservatives (e.g.,methyl or propyl p hydroxybenzoates or sorbic acid). The preparationscan also contain buffer salts, preservatives, flavoring, coloring andsweetening agents as appropriate.

Nutraceutical Composition Formulations and Methods of Preparation

Another aspect of the application relates to a method of preparing aMoMo30 containing nutraceutical composition or purified MoMo30 protein,including but not limited to plants of the Momordica genus, such asMomordica balsamina. In preferred embodiments, the MoMo30 protein isobtained from Momordica balsamina leaf extracts. In other embodiments,the nutraceutical composition is obtained from Momordica balsamina fruitextracts, root extracts, bark extracts, seed extracts and/or any flowerthereof.

In one embodiment, the method includes one or more steps including:harvesting the plants; drying the plants; extracting the dried plants inwater or aqueous media to form an aqueous extract; and centrifuging theaqueous extract to remove debris and particulates.

Given that MoMo30 retains its activity in a MoMo30-containing extractafter boiling, in some embodiments, the MoMo30 protein is purified byboiling the extract for 20 min to inactivate other proteins in theextract.

In some embodiments, the aqueous MoMo30 extract is passed through amolecular weight cutoff (MWCO) filter (e.g., Amicon 30 kDa or 50 kDa);the retentate containing MoMo30 protein is collected; the MoMo30 proteineluted from the retentate; drying the protein in the retentate orresuspending the protein in buffer for further analysis, purificationand/or storage. The MoMo30 protein may be further purified from theplant extract by immunoaffinity chromatography and other conventionalmethodologies known to those of skill in the art. In some embodiments,plant leaves comprising a MoMo30 protein are obtained from members ofthe Momordica genus, such as Momordica balsamina.

In a particular embodiment, a method for preparing a MoMo30-containingplant extract comprises the steps of: (a) drying a plant comprising anMoMo30 protein; (b) extracting the dried plant in aqueous media; (c)separating the aqueous media from solid material to form an aqueousMoMo30 extract; and (d1) passing the aqueous extract through a molecularweight cut-off filter and collecting a retentate comprising the protein,or (d2) purifying the protein from the aqueous extract by immunoaffinitypurification using an antibody directed against the protein.

In another embodiment, a method for preparing a MoMo30-containing plantextract comprises the steps of: (a) drying a plant comprising an MoMo30protein; (b) extracting the dried plant in boiling water; and (c)separating the aqueous media from solid material to form an aqueousMoMo30 extract.

The MoMo30 protein may be dried for storage or resuspended in anappropriate buffer for further use or storage following e.g.,quantification of MoMo30 yield and/or characterization of MoMo30 purity.In practice, the extracts are highly stable and have been stored freezedried for years without significant loss of anti-viral activity.

In addition, the extract, purified extract and/or purified MoMo30protein may be characterized by HPLC and/or tested for functionalactivity via infectivity assays and the like. For example, in someembodiments, the MoMo30-containing plant extract or purified protein maybe evaluated for functional activity by testing their ability to inhibitinfection by HIV using a MAGI cell infectivity assay (or “indicatorassay”). This assay involves the use of genetically modifiedCD4-expressing HeLa cell line (MAGI) containing an HIV LTR-drivencassette placed upstream of the E. coli β-gal encoded reporter gene(HeLa-CD4-LTR-β-gal). See Kimpton and Emerman, J. Virol., 66:2232, 1992.Expression of the reporter gene is activated in the presence of HIV Tat,which is expressed upon infection by HIV, such as HIV-1NL4-3 andactivates the HIV-1 LTR. Cells infected by HIV turn blue and can becounted under a microscope.

The nutraceutical compositions of the present application can bemanufactured by methods of conventional mixing, dissolving, granulating,dragee-making levigating, emulsifying, encapsulating, entrapping orlyophilization processes. The compositions can be formulated inconventional manner using one or more nutraceutically acceptablecarriers, including excipients, binders, diluents, antiadherents,coatings, disintegrants, flavors, colors, lubricants, glidants,sorbents, preservatives, sweeteners or auxiliaries that facilitateprocessing of the compositions into preparations that can be used.

Exemplary excipients include, but are not limited to binders, diluentsor fillers, such as dextrates, dicalcium phosphate, calcium sulfate,lactose, cellulose, kaolin, mannitol, sodium chloride, dry starch,sorbitol, sucrose, inositol, powdered sugar, bentonite, microcrystallinecellulose, hydroxypropylmethylcellulose. hypromellose, rice flour, ricebran oil, gelatin, vegetable glycerin, lecithin, magnesium stearate,cellulose, inulin, and silicon dioxide.

As used herein, the term “binder” refers to substances that are added topowdered particles, generally prior to granulation or directcompression, to achieve the requisite flow property and/orcompressibility necessary for effective compression of the powderyparticles and/or granules into a tablet, lozenge, or capsule, or toimprove certain physical properties of the powdered particles includingbut not limited to increasing the cohesive nature of the powderedparticles in forming granules, tablets, lozenges, capsules, and othersolid dosage forms.

Exemplary binders include, but are not limited to cellulose andcellulose derivatives (e.g. microcrystalline cellulose, methylcellulose(MC), sodium carboxymethyl cellulose (CMC), hydroxypropylmethylcellulose (HPMC, hypromellose), hydroxyethyl cellulose (HEC), andhydroxypropylcellulose (HPC); cellulose ethers; starch derivatives(including pregelatinized and granulated starches, dextrin, andmaltodextrin); arabogalactan; sugars (e.g., glucose, dextrose, lactose,and sucrose); sugar alcohols (e.g., mannitol, sorbitol, xylitol,erythritol, maltitol, and isomalt); polymers (e.g., likepolyvinylpyrrolidone (PVP, povidone), polyvinyl alcohol (PVA),polyacrylamides, poly-methyacrylamides, polyoxazolines (POZ),polyphosphates, and polyethylene glycol (PEG)); copolymers (e.g.,divinyl ether-maleic anhydride); vegetable waxes (e.g., camauba wax),gelatins and gelatin-like products (e.g., agar); pectins;oligosaccharides or polysaccharides (e.g., inulin and xanthan gum); anddietary fiber (e.g., chicory root and chicory root extracts); gelatin,molasses, acacia gum, panwar gum, ghatti gum, sodium alginate, Irishmoss extract, mucilage of isapgol husks, Veegum and combinationsthereof. Exemplary lubricants include, but not limited to, talc,magnesium stearate, calcium stearate, stearic acid, hydrogenatedvegetable oils, polyethylene glycol, sodium benzoate, sodium acetate,sodium chloride, leucine, carbowax, sodium lauryl sulfate, and magnesiumlauryl sulfate may be added to the hevamine A-related proteincomposition. Also, glidants, such as but not limited to, colloidalsilicon dioxide or talc may be added to improve the flow characteristicsof a powdered ingredient. Finally, disintegrants, such as but notlimited to, starches, clays, celluloses, algins, gums, crosslinkedpolymers (e.g., croscarmelose, crospovidone, and sodium starchglycolate), Veegum, methylcellulose, agar, bentonite, cellulose and woodproducts, natural sponge, cation-exchange resins, alginic acid, guargum, citrus pulp, carboxymethylcellulose, or sodium lauryl sulfate withstarch may also be added to facilitate disintegration of the activeingredients in the intestine.

In some embodiments, the nutraceutical composition is in dried form. Inparticular embodiments, the dried form is a powdered form, granulatedform, capsule, tablet, lozenge, or herbal tea extract.

In certain embodiments, the dried form is agitated or dissolved in aliquid, such as water, carbonated water, juice, herbal tea, soft drinks,energy drinks, or milk. The nutraceutical ingredients described hereinmay be powdered or granulated prior to being incorporated into thenutraceutical composition.

In certain embodiments, one or more of the nutraceutical ingredients maybe powdered or granulated to a particle size between about 10 micronsand about 300 microns, and more particularly between about 100 micronsand about 200 microns. However, one of ordinary skill in the art mayselect other suitable particle sizes, including particle sizes adaptedto facilitate water solubility of the nutraceutical compositions asfurther described herein, as desired. Furthermore, each of thesenutraceutical ingredients may be substantially evenly mixed togetheraccording to conventional techniques to provide the nutraceuticalcomposition for convenient end use.

In particular embodiments, the ingredients are provided in a dosage formsuitable for oral administration, including one or more tablets orartificial capsules, a manufactured or compounded liquid or slurry form,or as a manufactured powder or granulate.

As a nonlimiting example, the powder or granulate form of thenutraceutical formulation may be water soluble. In particular, thenutraceutical formulation may be ground to, or otherwise provided in, aparticle size that is adapted to naturally dissipate and dissolve withinan aqueous medium. It should be appreciated that where the powdered orgranulated ingredients of the nutraceutical formulation are dehydrated,the ingredients will furthermore more readily absorb water and dissolvein the aqueous medium, especially in comparison to synthetic vitaminalternatives. One of ordinary skill in the art may also select othersuitable dosage forms within the scope of the present disclosure.

It should be appreciated that the capsule dosage form for thenutraceutical formulation may be preferred. Where provided in a capsuledosage form, the artificial capsules may be single-piece or two-piecemanufactured bodies for encapsulation of the formulation. Suitableingredients for the manufactured capsules may include, but are notlimited to wax, cellulose (including, for example, Hypromellose or HPMC,and sometimes referred to as “veggie capsule”), starches, gelatin,pullulan/tapioca, and combinations thereof. Other suitable ingredientsfor capsules of the present disclosure may also be employed, as desired.

One of ordinary skill in the art may also select other suitable dosageforms and capsule types within the scope of the present application.

In some embodiments, a dried nutraceutical composition is enclosedwithin an edible film which dissolves upon agitation or contact with abeverage, such as water, carbonated water, juice, herbal tea, softdrinks, energy drinks, or milk. In some embodiments, the edible filmcomprises one or more ingredients selected from the group consisting ofpolyethylene oxide (PEO), pullulan, hydroxypropylmethyl cellulose(HPMC), hydroxyethyl cellulose (HPC), hydroxypropyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium aginate,polyethylene glycol, xanthan gum, tragancanth gum, guar gum, acacia gum,arabic gum, polyacrylic acid, methylmethacrylate copolymer, carboxyvinylcopolymers, starch, modified food starch, gelatin, ethyl cellulose,hydroxypropyl ethyl cellulose, cellulose acetate phthalate, orhydroxypropyl methyl cellulose phthalate, and combinations thereof.

In some embodiments, the method of encapsulating the nutraceuticalpowder compositions may include forming a cavity in the film; fillingthe cavity with the nutritional powder composition; and sealing thefilm. Another method of encapsulating the nutritional powder compositionmay include creating a back and bottom seal with the film; filling thefilm with the nutritional powder composition; and creating a top sealwith the film. Compositions and methods for enclosing nutritionalcompositions within an edible film are described in U.S. PatentPublication No. 2018/0290804, the disclosure of which is incorporated byreference in its entirety.

In some embodiments, the present application provides a liquidcontaining a dried form of the nutraceutical composition in a beverage,such as water, juice, herbal tea, soft drinks, energy drinks, or milk.In certain embodiments, the beverage provides one or more natural orsynthetic sweeteners.

In certain embodiments, the natural sweetener is a high intensitysweetener selected from one or more Stevia extracts (i.e., from Steviarebaudiana) and steviol glycosides therefrom including e.g.,rebaudioside A and rebaudioside D; monk fruit extracts (from Siraitiagrosvenorii) and mogrosides therefrom; sweet tea extracts (from Rubussuavissimus) and suaviosides therefrom; and combinations thereof.

In other embodiments, the beverage provides one or more natural orsynthetic sweeteners selected from the group consisting of aspartame(e.g., NutraSweet), sucralose (Splenda), acesulfame potassium (alsoknown as acesulfame K, or Ace-K), advantame, sorbitol, xylitol,mannitol, neotame, erythritol, trehalose, raffinose, cellobiose,tagatose, allulose, inulin,N—[N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-alpha-aspartyl]-L-phenylalanine1-methyl ester, glycyrrhizin, sodium cyclamate, brazzein, miraculin,curculin, pentadin, mabinlin, NHDC, thaumatin, naringin dihydrochalcone,maltol, ethyl maltol, advantame, and combinations thereof.

Liquid preparations for oral administration can take the form of, forexample, solutions, elixirs, syrups or suspensions, or they can bepresented as a dry product for constitution with water or other suitableliquids described herein before use. Such liquid preparations can beprepared by conventional means with nutraceutically acceptable additivessuch as suspending agents (e.g., sorbitol syrup, cellulose derivativesor hydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non aqueous vehicles (e.g., almond oil, oily esters, ethylalcohol, or fractionated vegetable oils); and preservatives (e.g.,methyl or propyl p hydroxybenzoates or sorbic acid). The preparationscan also contain buffer salts, preservatives, flavoring, coloring andsweetening agents as appropriate.

In some embodiments, the nutraceutical composition includes a MoMo30protein expressed from an expression vector by recombinant DNAtechnology.

An “expression vector” is used herein with reference to a non-viral orviral vector containing a polynucleotide encoding an antimicrobialMoMo30 protein of the present application in a form suitable forexpression of MoMo30 in a host cell. The expression vectors include oneor more regulatory sequences, selected based on the host cells used forexpression, and operably linked to the polynucleotide sequence to beexpressed. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, and the like.

As used herein, the term “control sequences” or “regulatory sequences”refers to DNA sequences necessary for the expression of an operablylinked coding sequence in a particular host organism. The term“control/regulatory sequence” is intended to include promoters,enhancers, and other expression control elements (e.g., polyadenylationsignals). Control/regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcells and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Anexpression vector may be designed to facilitate expression of anantimicrobial MoMo30 protein-encoding polynucleotide in one or more celltypes. Tissue-specific regulatory elements may be used to restrictexpression to a particular cell type.

A nucleic acid sequence is “operably linked” to another nucleic acidsequence when the former is placed into a functional relationship withthe latter. For example, a DNA for a presequence or secretory leaderpeptide is operably linked to DNA for a protein if it is expressed as apreprotein that participates in the secretion of the protein; a promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. By contrast, enhancers need not be contiguous with e.g.,promoters. As used herein, the term “preprotein” is used with referenceto a predicted amino acid sequence including an N-terminal signalpeptide, which is cleaved off during protein processing resulting in asecreted biologically active mature protein as described herein.

As used herein, the term “promoter” related to an expression controlelement for a structural gene and to which RNA polymerase specificallybinds and initiates RNA synthesis (transcription) of that gene. The termpromoter is to be taken in its broadest context and includestranscriptional regulatory elements (TREs) from genomic genes orchimeric TREs therefrom, including the TATA box or initiator element foraccurate transcription initiation, with or without additional TREs(i.e., upstream activating sequences, transcription factor bindingsites, enhancers and silencers) which regulate activation or repressionof genes operably linked thereto in response to developmental and/orexternal stimuli and trans-acting regulatory proteins or nucleic acids.The promoter may be constitutively active, or it may be active in one ormore tissues or cell types in a developmentally regulated manner. Apromoter may contain a genomic fragment, or it may contain a chimera ofone or more TREs combined.

Examples of promoters include: the immediate early promoter of CMV, LTRor SV40 promoter, polyhedron promoter of baculovirus, E. coli lac or trppromoter, phage T7 and lambda PL promoter and other promoters known tocontrol expression of genes in prokaryotic or eukaryotic cells or theirviruses. The expression vector typically also contains a ribosomebinding site for translation initiation and a transcription terminator.The vector includes appropriate sequences for amplifying expression. Inaddition, the expression vectors comprise one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells, such as dihydrofolate reductase or neomycin resistance foreukaryotic cell culture or such as tetracycline or ampicillin resistancein E. coli.

The expression vector can also include additional expression elements,for example, to improve the efficiency of translation. These signals caninclude, e.g., an ATG initiation codon and adjacent sequences. In somecases, for example, a translation initiation codon and associatedsequence elements are inserted into the appropriate expression vectorsimultaneously with the polynucleotide sequence of interest (e.g., anative start codon). In such cases, additional translational controlsignals are not required. However, in cases where only a protein codingsequence or a portion thereof, is inserted, exogenous translationalcontrol signals, including an ATG initiation codon is provided forexpression of an antimicrobial MoMo30 protein. The initiation codon isplaced in the correct reading frame to ensure translation of thepolynucleotide sequence of interest. Exogenous transcriptional elementsand initiation codons can be of various origins, both natural andsynthetic. If desired, the efficiency of expression can be furtherincreased by the inclusion of enhancers appropriate to the cell systemin use.

Expression vectors carrying an antimicrobial MoMo30-encoding nucleicacid can be introduced into host cells by any of a variety of well-knownprocedures, such as electroporation, calcium phosphate precipitationliposome mediated transfection, infection, transfection, and the like,depending on the selection of vectors and host cells.

Host cells that contain antimicrobial MoMo30 protein-encoding nucleicacids are, thus, also a feature of this disclosure. Favorable host cellsinclude prokaryotic (i.e., bacterial) host cells, such as E. coli, aswell as numerous eukaryotic host cells, including plant (e.g., tobacco),fungal (e.g., yeast, such as Saccharomyces cerevisiae and Picchiapastoris) cells, insect cells, and mammalian cells (such as CHO cells).Recombinant antimicrobial MoMo30-encoding nucleic acids are introduced(e.g., transduced, transformed, or transfected) into host cells, forexample, via a vector, such as an expression vector. As described above,the vector is most typically a plasmid, but such vectors can also be,for example, a viral particle, a phage, etc. Examples of appropriateexpression hosts include bacterial cells, such as E. coli, Streptomyces,and Salmonella typhimurium; fungal cells, such as Saccharomycescerevisiae, Pichia pastoris and Neurospora crassa; insect cells such asDrosophila and Spodoptera frugiperda; mammalian cells such as 3T3, COS,CHO, BHK, HEK 293 or Bowes melanoma; plant cells, including algae cells,etc.

The host cells can be cultured in conventional nutrient media modifiedas appropriate for activating promoters, selecting transformants oramplifying the inserted polynucleotide sequences. The cultureconditions, such as temperature, pH, and the like, are typically thosepreviously used with the host cell selected for expression and will beapparent to those skilled in the art.

In bacterial systems, a number of expression vectors can be selecteddepending upon the use intended for the expressed product. For example,when large quantities of a protein or fragments thereof are needed forthe production of antibodies, vectors which direct high-level expressionof fusion proteins that are readily purified are favorably employed.Such vectors include, but are not limited to, multifunctional E. colicloning and expression vectors such as BLUESCRIPT (Stratagene), in whichthe coding sequence of interest, e.g., a polynucleotide of the inventionas described above, can be ligated into the vector in-frame with e.g.,sequences for the amino-terminal translation initiating methionine andthe subsequent 7 residues of beta-galactosidase producing acatalytically active beta galactosidase fusion protein in which theamino-terminal methionine is ligated in frame with a histidine tag; andthe like.

Similarly, in yeast, such as Saccharomyces cerevisiae, a number ofvectors containing constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH can be used for production of thedesired expression products. In mammalian host cells, a number ofexpression systems, including both plasmids and viral-based systems, canbe utilized.

A host cell is chosen for its ability to modulate the expression of theinserted sequences or to process the expressed protein in the desiredfashion. Such modifications of the protein include, but are not limitedto, glycosylation, acetylation, carboxylation, phosphorylation,lipidation, acylation etc. Post-translational processing for example,which cleaves a precursor form into a mature form of the protein (forexample, by a furin protease) is performed in the context of the hostcell. Different host cells such as 3T3, COS, CHO, HeLa, BHK, MDCK, 293,W138, etc. have specific cellular machinery and characteristicmechanisms for such post-translational activities and can be chosen toensure the correct modification and processing of the introduced,foreign protein.

For long-term, high-yield production of recombinant antimicrobial MoMo30protein, stable expression systems may be employed. For example,polynucleotides encoding an antimicrobial MoMo30 protein can beintroduced into suitable host cells using expression vectors whichcontain viral origins of replication or endogenous expression elementsand a selectable marker gene. Following the introduction of the vector,cells are allowed to grow for 1-2 days in an enriched media before theyare switched to selective media. The purpose of the selectable marker isto confer resistance to selection and its presence allows growth andrecovery of cells which successfully express the introduced sequences.For example, resistant groups or colonies of stably transformed cellscan be proliferated using tissue culture techniques appropriate to thecell type. Host cells transformed with a nucleic acid encoding anantimicrobial MoMo30 protein are cultured under conditions suitable forthe expression and recovery of the encoded protein from cell culture.

Following transduction of a suitable host cell line and growth of thehost cells to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period. The secretedprotein product is then recovered from the culture medium.Alternatively, cells can be harvested by centrifugation, disrupted byphysical or chemical means and the resulting crude extract retained forfurther purification. Eukaryotic or microbial cells employed inexpression of proteins can be disrupted by any convenient method,including freeze-thaw cycling, sonication, mechanical disruption or useof cell lysing agents or other methods, which are well known to thoseskilled in the art.

Expressed antimicrobial MoMo30 proteins can be recovered and purifiedfrom recombinant cell cultures by any of a number of methods well knownin the art, including ammonium sulfate or ethanol precipitation, acidextraction, anion, or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g., using any of the tagging systems noted herein),hydroxylapatite chromatography and lectin chromatography. Since theMoMo30 protein is unusually heat stable it also suggests thatapplication of heat to denature other proteins may be a useful approach.Protein refolding steps can be used, as desired, in completingconfiguration of the mature protein. Finally, high performance liquidchromatography (HPLC) can be employed in the final purification steps.

In some embodiments, the MoMo30-encoded expression vector is introducedinto a suitable bacterial host. Numerous suitable strains of E. coli areavailable and can be selected by one of skill in the art (for example,the Rosetta and BL21 (DE3) strains).

In other embodiments, the MoMo30-encoded expression vector is introducedinto insect cells using a baculovirus expression vector system (BEVS).Recombinant baculovirus capable of infecting insect cells can begenerated using commercially available vectors, kits and/or systems,such as the BD BaculoGold system from BD BioScience. Briefly, thepolynucleotide sequence encoding the antimicrobial product is insertedinto the pAcSG2 transfer vector. Then, host cells SF9 (Spodopterafrugiperda) are co-transfected by pAcSG2-chimer plasmid and BDBaculoGold, containing the linearized genomic DNA of the baculovirusAutographa californica nuclear polyhedrosis virus (AcNPV). Followingtransfection, homologous recombination occurs between the pACSG2 plasmidand the Baculovirus genome to generate the recombinant virus. In oneexample, the antimicrobial product is expressed under the regulatorycontrol of the polyhedrin promoter (pH). Similar transfer vectors can beproduced using other promoters, such as the basic (Ba) and p10promoters. Similarly, alternative insect cells can be employed, such asSF21 which is closely related to the SF9 and the High Five (Hi5) cellline derived from a cabbage looper, Trichoplusia ni.

Following transfection and induction of expression (according to theselected promoter and/or enhancers or other regulatory elements), theexpressed proteins are recovered (e.g., purified or enriched) andrenatured to ensure folding into a biologically active conformation.

Methods of Using the Nutraceutical Composition

In another aspect, the present application provides a method forpreventing or treating a microbial infection. In one embodiment, themethod comprises orally administering to a subject a nutraceuticalcomposition comprising MoMo30 protein alone or in a combinationformulation with or without additional nutraceutical ingredients asdescribed above in an amount sufficient to prevent a microbialinfection, reduce the symptoms associated with the infection, or curethe subject of the microbial infection or disease. The nutraceuticalcomposition may be administered as a MoMo30 protein or MoMo30 extractalone or in combination with other nutritional ingredients, plantextracts or plant components described above.

In some embodiments, the nutraceutical composition is administered in adried form, such as a capsule, tablet, lozenge, or powder with at leastone nutraceutically acceptable carrier.

In some embodiments, the nutraceutical composition is administered as aliquid or beverage with or without one or more other nutraceuticals. Theliquid may be, for example, a beverage, such as water, juice, herbaltea, soft drink, energy drink, milk, and others known in the art.

In one embodiment, the nutraceutical composition is administered as oneor more herbal extracts derived from a natural plant source, such as M.balsamina. Further, plant extracts may be used to isolate purified orsemi-purified MoMo30 protein or any of the plant nutraceuticalingredients described herein.

In some embodiments, the composition is used to prevent or treat aninfection caused by a virus.

In other embodiments, the composition is used to prevent or treat aninfection caused by a bacterium.

In other embodiments the composition is used to prevent or treat aninfection caused by a fungus.

In other embodiments, the composition is used to prevent or treat aninfection caused by a protozoa.

In certain preferred embodiments, the virus, bacterium, fungus, orprotozoa includes one or more cell surface proteins containing one ormore sugar residues, such as mannose, sialic acid, glucose, glucuronicacid, xylose, fucose, galactose, N-acetylglucosamine,N-acetylgalactosamine, and/or iduronic acid.

Exemplary viruses for prevention or treatment include a variety ofenveloped RNA and DNA viruses, including RNA viruses, such asretroviruses, lentiviruses, coronaviruses (including subgroup 1a and 1balphacoronaviruses, subgroup 2a, 2b, 2c and 2d betacoronaviruses, andsubgroup 3 gammacoronaviruses), herpesviruses, alphaviruses,bunyaviruses, filoviruses, flaviviruses, hepatitis viruses,orthomyxoviruses (e.g., influenza Types A, -B, -C, -D), paramyxoviruses,rhabdoviruses, and togaviruses; and DNA viruses, such as herpesviruses,poxviruses, and hepadnaviruses. In certain preferred embodiments, theinfection is caused by HIV, SARS-CoV-2, or an influenza Type 1 virus.

Exemplary species of enveloped viruses for prophylactic or therapeuticuse include retroviruses or lentiviruses, such as human immunodeficiencyvirus type 1 and type 2 (HIV-1 and HIV-2), human T-cell lymphotropicvirus type I and type II (HTLV-I and HTLV-II); herpesviruses, includingEpstein-Barr virus, human cytomegalovirus type 1 (HCMV-1), human herpesvirus type 6 (HHV-6), human herpes virus type 7 (HHV-7), human herpesvirus type 8 (HHV-8), influenza type A virus, including subtypes H1N1and H5N1, as well as types -B, -C, and -D; coronaviruses, includingsevere acute respiratory syndrome coronavirus type 2 (SARS-CoV-2),SARS-CoV-1, Middle East Respiratory Syndrome Coronavirus (MERS-CoV),HCoV-229E, HCoV-0C43, HCoV-NL63, and HCoV-HKU1; RNA viruses that causehemorrhagic fever, such as the filoviruses, Ebola virus (EBOV) andMarburg virus (MBGV); Bunyaviridae (e.g., Rift Valley fever virus (RVFV)and Crimean-Congo hemorrhagic fever virus (CCHFV)); and flaviviruses,such as Hepatitis C virus, West Nile virus (WNV), Dengue fever virus(DENV), yellow fever virus (YFV), tick-borne encephalitis virus, SaintLouis encephalitis virus, and (GB virus C (GBV-C), formerly known asHepatitis G virus (HGV)); enteroviruses (Types A to L, includingcoxsackieviruses (Types A to C); echoviruses; rhinoviruses (Types A toC), poliovirus); orthomyxoviruses (e.g., influenza Types A, -B, -C, -D,including A subtypes H1N1, H5N1, H3N2); paramyxoviruses (e.g.,rubulavirus (mumps), rubeola virus (measles), respiratory syncytialvirus, Newcastle disease, parainfluenza); parvoviruses (e.g., parvovirusB19 virus); rhabdoviruses (e.g., Rabies virus); arenaviruses (e.g.,lymphocytic choriomeningitis virus and several Lassa fever viruses,including Guanarito virus, Junin virus, Lassa virus, Lujo virus, Machupovirus, Sabia virus, Whitewater Arroyo virus); alphaviruses (e.g.,Venezuelan equine encephalitis virus, eastern equine encephalitis virus;western equine encephalitis virus); hepatitis A virus, hepatitis C virus(HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), including anytype, subtype, clade or sub-clade of the foregoing viruses.

In certain preferred embodiments, the RNA virus for prevention ortreatment is a coronavirus, such as SARS-CoV-2, SARS-CoV-1, MERS-CoV,HCoV-229E, HCoV-0C43, HCoV-NL63, and HCoV-HKU1. In an exemplaryembodiment, a method for preventing or reducing symptoms of acoronavirus infection, comprises orally administering to a subject inneed thereof a composition comprising: an effective amount of a MoMo30protein comprising an amino acid sequence that is at least 90% identicalto SEQ ID NO: 4; and at least one nutraceutically acceptable carrier.

In other preferred embodiments, the RNA virus for prevention ortreatment is an influenza Type A virus. Influenza A viruses are dividedinto subtypes on the basis of two proteins on the surface of the virus,hemagglutinin (HA) and neuraminidase (NA). There are 18 known HAsubtypes and 11 known NA subtypes. Many different combinations of HA andNA proteins are possible. For example, an “H7N2 virus” designates aninfluenza A virus subtype that has an HA7 protein and an NA2 protein.Similarly, an “H5N1” virus has an HA5 protein and an NA1 protein. Type Ainfluenza viruses that may be targeted for prophylactic and/ortherapeutic use according to the methods and compositions of the presentapplication include a variety of sub-types, such as H1N1, H1N2, H3N2,H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, and H5N9, H7N1, H7N2,H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, H7N9, H9N1, H9N2, H9N3, H9N4, H9N5,H9N6, H9N7, H9N8, H9N9, H17N10 and H18N11).

In another exemplary embodiment, a method for preventing or reducingsymptoms of an influenza Type A virus infection, comprises orallyadministering to a subject in need thereof a composition comprising: aneffective amount of a MoMo30 protein comprising an amino acid sequencethat is at least 90% identical to SEQ ID NO: 4; and at least onenutraceutically acceptable carrier.

Exemplary species of enveloped DNA viruses for prevention or treatmentinclude, but are not limited to, Exemplary DNA viruses for prophylacticor therapeutic treatment include herpesviruses (e.g., HSV-1, HSV-2, EBV,VZV, HCMV-1, HHV-6, HHV-7, HHV-8), papillomaviruses (e.g., humanpapilloma virus (HPV) Types 1, 2, 4, 6, 11, 16, 18, 26, 30, 31, 33, 34,35, 39, 40, 41, 42, 43, 44, 45, 51, 52, 54, 55, 56, 57, 58, 59, 61, 62,64, 67, 68, 69, 70); poxviruses (e.g., smallpox virus), hepadnaviruses(Hepatitis B virus); anelloviruses (e.g., transfusion transmitted virusor torque teno virus (TTV); as well as any type, subtype, clade orsub-clade thereof.

In some embodiments, the MoMo30 protein is used for the treatment orprevention of bacterial infection. Exemplary bacteria for treatmentinclude, but are not limited to, Staphylococcus species, including S.epidermidis, S. aureus, and methicillin-resistant S. aureus;Enterococcus species, including E. faecalis and E. faecium;Mycobacterium tuberculosis, Haemophilus influenzae, Pseudomonas species,including P. aeruginosa, P. pseudomallei, and P. mallei; Salmonellaspecies, including S. enterocolitis, S. typhimurium, S. enteritidis, S.bongori, and S. choleraesuis; Shigella species, including S. flexneri,S. sonnei, S. dysenteriae, and S. boydii; Brucella species, including B.melitensis, B. suis, B. abortus, and B. pertussis; Neisseria species,including N. meningitidis and N. gonorrhoeae; Escherichia coli,including enterotoxigenic E. coli (ETEC); Vibrio cholerae, Helicobacterpylori, Chlamydia trachomatis, Clostridium difficile, Cryptococcusneoformans, Moraxella catarrhalis, Campylobacter species, including C.jejuni; Corynebacterium species, including C. diphtheriae, C. ulcerans,C. pseudotuberculosis, C. pseudodiphtheriticum, C. urealyticum, C.hemolyticum, C. equi; Streptococcus species, including S. pneumoniae, S.pyogenes, S. mutans, S. agalactiae, S. equi, S. canis, S. bovis, S.equinus, S. anginosus, S. sanguis, S. salivarius, S. mitis; Listeriamonocytogenes, Nocardia asteroides, Bacteroides species, Actinomycetesspecies, Treponema pallidum, Leptospirosa species, Klebsiellapneumoniae; Proteus sp., including Proteus vulgaris; Serratia species,Acinetobacter, Yersinia species, including Y. pestis and Y.pseudotuberculosis; Francisella tularensis, Enterobacter species,Bacteroides species, Legionella species, Borrelia burgdorferi, and thelike.

In some embodiments, the MoMo30 protein is used for the treatment orprevention of a fungal infection. Exemplary fungi for treatment include,but are not limited to, Aspergillus species, Dermatophytes, Blastomycesderinatitidis, Candida species, including C. albicans and C. krusei;Malassezia furfur, Exophiala werneckii, Piedraia hortai, Trichosporonbeigelii, Pseudallescheria boydii, Madurella grisea, Histoplasmacapsulatum, Sporothrix schenckii, Histoplasma capsulatum, Tinea species,including T. versicolor, T. pedis, T. unguium, T cruris, T. capitus, T.corporis, T. barbae; Trichophyton species, including T. rubrum, T.interdigitale, T. tonsurans, T. violaceum, T. yaoundei, T. schoenleinii,T. megninii, T. soudanense, T. equinum, T. erinacei, and T. verrucosum;Microsporum species, including M. audouini, M. ferrugineum, M. canis, M.nanum, M. distortum, M. gypseum, M. fulvum, and the like.

In certain embodiments, the MoMo30 protein may be useful for preventingor treating a variety of conditions including, for example, infectionsof the skin, infections of the urogenital tract, infections of thedigestive system (e.g., the gut), infections of the lung, and/orinfections of the sinus. For example, the antimicrobial compositions maybe useful for the treatment of a condition, such as, for example,rosacea, atopic dermatitis (e.g., eczema), a Candida infection (e.g.,vaginal, diaper, intertrigo, balanitis, oral thrush), Tinea versicolor,Dermatophytosis (e.g., Tinea pedis (athlete's foot)), Tinea unguium,Onychomycosis (e.g., toe nail fungus), Tinea cruris, Tinea capitus,Tinea corporis, Tinea barbae, seborrheic dermatitis,antibiotic-resistant skin infections, impetigo, ecthyma, erythrasma,burn wounds (e.g., reduction of infections, improved healing), diabeticfoot/leg ulcers (e.g., reduction of infections, improved healing),prevention of central catheter-related blood stream infections, oralmucositis, warts (e.g., common, flat, plantar, genital), and molluscumcontagiosum. In some embodiments, the condition is acne, often acnevulgaris and sometimes acne conglobate.

In some embodiments, the MoMo30 protein may be useful for treating orpreventing a protozoan infection. Exemplary protozoan infectionsinclude, but are not limited to those caused by Cryptosporidium,Isospora belli, Toxoplasma gondii, Trichomonas vaginalis, and Cyclosporaspecies.

Administration of Nutraceutical Composition

The nutraceutical ingredients of the present application are generallyintroduced with one or more nutraceutically acceptable carriers. A“nutraceutically acceptable carrier” is intended to include any and allsolvents, solubilizers, fillers, stabilizers, binders, absorbents,bases, buffering agents, lubricants, controlled release, vehicles,diluents, emulsifying agents, humectants, lubricants, dispersion media,coatings, antibacterial or antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with nutraceuticaladministration. The use of such media and agents for nutraceuticallyactive substances is well-known in the art. Except as far as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary agentscan also be incorporated into the compositions. In certain embodiments,the nutraceutically acceptable carrier comprises serum albumin.

In preferred embodiments, the nutraceutical composition is orallyadministered. Oral compositions generally include an inert diluent or anedible carrier. They can be enclosed in gelatin capsules or compressedinto tablets. For oral therapeutic administration, the active compoundmay be incorporated with excipients and used in the form of tablets,troches, or capsules. Nutraceutically compatible binding agents, and/oradjuvant materials can be included as part of the composition. Thetablets, pills, capsules, lozenges, and the like can contain any of thefollowing ingredients, or compounds of a similar nature: a binder suchas microcrystalline cellulose, gum tragacanth or gelatin; an excipientsuch as starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orStertes; a glidant such as colloidal silicon dioxide; a sweetening agentsuch as sucrose or saccharin; or a flavoring agent such as peppermint,methyl salicylate, or orange flavoring.

For administration by inhalation, the nutraceutical ingredients may bedelivered in the form of an aerosol spray from pressured container ordispenser which contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer.

In some embodiments, local administration of the nutraceuticalcompositions of the present application may be carried out by topicaladministration.

Systemic administration can also be administered by transmucosal andtransdermal administration. Penetrants appropriate to the barrier to bepermeated may be used in such formulations. Such penetrants are known inthe art, and include, for example, for detergents, bile salts, andfusidic acid derivatives. In some embodiments, transmucosaladministration is accomplished using nasal sprays or suppositories. Fortransdermal administration, the nutraceutical compositions may beformulated into ointments, salves, gels, or creams as generally known inthe art.

In certain embodiments, the nutraceutical composition is formulated forsustained or controlled release of the active ingredients.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and poly lactic acid. Methods for preparation of suchformulations are well known to those skilled in the art. The materialscan also be obtained commercially from e.g., Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as nutraceutically acceptable carriers.

It is especially advantageous to formulate the nutraceutical compositionin dosage unit form for ease of administration and uniformity of dosage.Suitable unit dosage forms include, but are not limited to powders,tablets, pills, capsules, liquids, teas, lozenges, suppositories,patches, nasal sprays, lipid complexes, etc.

A dosage unit form as used herein includes physically discrete unitssuited as unitary dosages for the subject; each unit containing apredetermined quantity of nutraceutical ingredients determined toproduce the desired therapeutic effect in association with the anynutraceutically acceptable carrier(s). The specific dosage unit forms ofthe present application may be dictated by or directly dependent on theunique characteristics of the nutraceutical ingredients and theparticular effects to be achieved, and the limitations inherent in theart of compounding such an active compound for the treatment ofindividuals. The nutraceutical composition according to the presentapplication may be administered orally, sublingually, topically ortransmucosally.

As a general proposition, the nutraceutical ingredient(s) areadministered or formulated for administration, in a weight range ofabout 1 ng/kg body weight/day to about 100 mg/kg body weight/day whetherby one or more administrations. In more particular embodiments, thenutraceutical ingredient(s) are administered or formulated foradministration, in a weight range from about 1 ng/kg body weight/day toabout 1 μg/kg body weight/day, 1 ng/kg body weight/day to about 100ng/kg body weight/day, 1 ng/kg body weight/day to about 10 ng/kg bodyweight/day, 10 ng/kg body weight/day to about 1 μg/kg body weight/day,10 ng/kg body weight/day to about 100 ng/kg body weight/day, 100 ng/kgbody weight/day to about 1 μg/kg body weight/day, 100 ng/kg bodyweight/day to about 10 μg/kg body weight/day, 1 μg/kg body weight/day toabout 10 μg/kg body weight/day, 1 μg/kg body weight/day to about 100μg/kg body weight/day, 10 μg/kg body weight/day to about 100 μg/kg bodyweight/day, 10 μg/kg body weight/day to about 1 mg/kg body weight/day,100 μg/kg body weight/day to about 10 mg/kg body weight/day, 1 mg/kgbody weight/day to about 100 mg/kg body weight/day and 10 mg/kg bodyweight/day to about 100 mg/kg body weight/day.

In other embodiments, the nutraceutical ingredient(s) are administeredor formulated for administration, individually or collectively, at adosage range of 1 ng-10 ng per dose, 10 ng-100 ng per dose, 100 ng-1 μgper dose, 1 μg-10 μg per dose, 10 μg-100 μg per dose, 100 μg-1 mg perdose, 1 mg-10 mg per dose, 10 mg-100 mg per dose, and 100 mg-1000 mg perdose. The MoMo30 protein or MoMo30-containing formulation may beinjected once daily, twice daily, three times daily, and/or every 2, 3,4, 5, 6 or 7 days. In addition, the MoMo30 protein or MoMo30-containingformulation may be administered over a period of one month, two months,six months, 12 months, 2 years, 5 years, 10 years, 20 years, or more.

In other embodiments, the nutraceutical ingredient(s) are administered,individually or collectively, in a range from about 1 ng/kg to about 100mg/kg. In more particular embodiments, the nutraceutical ingredient(s)are administered or formulated for administration, individually orcollectively, in a dosage range from about 1 ng/kg to about 10 ng/kg,about 10 ng/kg to about 100 ng/kg, about 100 ng/kg to about 1 μg/kg,about 1 μg/kg to about 10 μg/kg, about 10 μg/kg to about 100 μg/kg,about 100 μg/kg to about 1 mg/kg, about 1 mg/kg to about 10 mg/kg, about10 mg/kg to about 100 mg/kg, about 0.5 mg/kg to about 30 mg/kg, andabout 1 mg/kg to about 15 mg/kg.

In other particular embodiments, the nutraceutical ingredient(s) may beadministered or formulated for administration, individually orcollectively, in a dose of 0.0006, 0.001, 0.003, 0.006, 0.01, 0.03,0.06, 0.1, 0.3, 0.6, 1, 3, 6, 10, 30, 60, 100, 300, 600 or 1000 mg/day.

For prophylactic or therapeutic use, the nutraceutical composition maybe administered once, twice, three times or four times per day with thedoses given at equal intervals throughout the day and night to maintaina constant presence of the drug to provide sufficient antimicrobialactivity.

In some embodiments, the treatment may be carried out for as long aperiod as necessary, i.e., until the infection is cleared or no longer athreat to the host. In some cases, the treatment may be continuedindefinitely while the disease state persists, although discontinuationmight be indicated if the antimicrobial compositions no longer produce abeneficial effect. For example, in some instances the treatment may becarried out for 1 month, 2 months, 4 months or 6 months and thendiscontinued.

II. Treatment of Plants with the Hevamine A-Related Composition

In another aspect, the present application provides a hevamine A-relatedcomposition for plant disease control, prevention or treatment. As usedherein, the term “plant” includes whole plants and parts thereof,including, but not limited to, shoot vegetative organs/structures (e.g.,leaves, stems and tubers), roots, flowers and floral organs/structures(e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules),seed (including embryo, endosperm, and seed coat) and fruit (the matureovary), plant tissue (e.g., vascular tissue, ground tissue, and thelike) and cells (e.g., guard cells, egg cells, and the like), andprogeny of same.

In one embodiment, plant disease control, prevention or treatment isaccomplished by applying an effective amount of the hevamine A-relatedcomposition either pre-or post-infection, to the whole plant or aportion of the plant such as the roots, stems, foliage, fruit, seeds,tubers or bulbs, or to the media (e.g., soil, sand or water) in whichthe plants to be protected are growing. In one aspect, the hevamineA-related protein is translocated through the vascular system in plantsand therefore the entire plant is not required to be contacted. Thus, inone aspect a portion of a plant may be treated with a hevamine A-relatedcomposition so that a plant disease is prevented, treated, or controlledin the treated portion, as well as in untreated portions of the plant,such as untreated leaves, stems, or roots.

In one embodiment, untreated leaves of wheat plants have decreaseddisease infection when lower leaves are treated with hevamine A-relatedcomposition. In another embodiment, disease control, prevention ortreatment corresponds to the concentration of MoMo30 protein in thetissue of the untreated leaf. In another embodiment, the hevamineA-related composition is be applied to the seed to protect the seed andseedling.

In one embodiment, a plant or plant part is contacted with the hevamineA-related composition either directly on a crop plant, or immediatelyadjacent to the crop plant where the MoMo30 protein can be taken-up intothe crop plant's vascular system. In methods where the composition isdirectly contacted with the crop plant, the composition may be contactedwith the entire crop plant or with only a portion of the plant.Additionally, a plant pathogen may be contacted with the hevamineA-related composition by e.g., direct contact on a plant surface. In apreferred aspect, a plant is contacted with the hevamine A-relatedcomposition by overhead spraying of the composition. Exemplary plantparts include leaves, roots, stems, fruit, seeds, tubers, bulbs, seeds,pollen, ovules, flowers, pods, stems, shoots, and combinations thereof.

Application of the hevamine A-related composition to the foliage ofplants is preferably accomplished by spraying, using any conventionalmeans for spraying liquids, such as spray nozzles or spinning-diskatomizers. The hevamine A-related composition is preferably diluteenough to be readily sprayed using standard agricultural sprayequipment. Suitable application rates for the present invention varydepending upon a number of factors, including e.g., the concentration ofMoMo30 protein and the plant species involved. Useful rates for applyingan aqueous composition to a field of foliage can range from about 25 toabout 1,000 liters per hectare (1/ha), preferably about 50 to about3001/ha, by spray application.

Suitable carriers for MoMo30 treatment of plants or plant parts includewater, aqueous solution, slurries, granules, or powders.

The plant treatment methods of the present invention find use in thecontrol, prevention or treatment of a wide variety of plant pathogens.The treatment methods include prophylactic inhibition and therapeutictreatment of infection by plant pathogens. Preferably, the methods ofthe present invention inhibit or treat plant pathogenic fungi, bacteriaand viruses, including any of those described herein.

Plant pathogens can be classified by their life cycle in relation to aplant host, these classifications include obligate parasites,facultative parasites, and facultative saprophytes. Obligate parasitescan only survive and reproduce by obtaining nutrition from living plantcells and are in direct contact with these cells. Examples of obligatefungal parasites of plants include, but are not limited to members ofUredinales (rusts), Ustilaginales (smuts and bunts), Erysiphales(powdery mildews), and Oomycetes (water molds and downy mildews).Facultative parasites are organisms that generally survive assaprophytes on the products of other organisms or dead organisms but canbecome parasitic when the conditions are favorable. Facultativesaprophytes are organisms that generally survive as parasites of plantsbut can survive as saprophytes when a susceptible plant host is notavailable.

In particular embodiments, the hevamine A-related compositions fordisease control is applied to the crop plant at a later growth stage,for example, when the plant is flowering or in the process of producingseeds or fruit, it is at these stages of development that plant diseasescan have the greatest effect on crop yield. Leaves are the sourcetissues that provide the products of photosynthesis needed for plantgrowth, seed, fruit and storage organ development. Protecting theseleaves from disease due to fungal infection is important to protectyield of the crop. The flag leaf of monocot crops contributessubstantially to the yield of the crop, protecting this leaf fromdisease is particularly important in protecting monocot crop yield.Leaves of dicot crops generally provide the products of photosynthesisto the closely associated fruiting structures of the plant, protectingthese leaves from disease is particularly important in protecting dicotcrop yields. Roots provide water and mineral nutrients to the plants,protecting roots from disease is also particularly important inmaintaining yield of the crop plant.

Enhanced formulations for systemic (includes both locally systemic andwhole plant systemic) uptake may include the addition of adjuvants, forexample, alkoxylated fatty amines, organosilicones, nonyl phenolethylene oxide condensate, and others known in the art. Examples ofsuitable adjuvants that enhance the uptake and efficacy of glyphosateinclude polyoxyalkylene alkylamines, polyoxyalkylene alkylammoniumsalts, polyoxyalkylene alkylamine oxides, polyoxyalkylene tertiary andquaternary etheramines, polyoxyalkylene etheramine oxides, mono- anddi-(polyoxyalkylene alcohol) phosphates, polyoxyalkylene alkylethers andcombinations thereof. Preferred adjuvants are polyoxyethylene coco andtallow amines, polyoxyethylene C8-18 alkyl oxypropyl amines,polyoxyethylene C16-22 alkylethers and combinations thereof. Examples ofthese adjuvants can be found in U.S. Pat. Nos. 5,668,085, 5,683,958,5,703,015, 6,063,733, 6,121,199, 6,121,200, 6,184,182, 6,245,713,6,365,551, RE37,866 and U.S. Patent Application Pub. No. US2003/0104943A1 (all of which are herein incorporated by reference in theirentirety).

In some embodiments, the hevamine A-related composition is applied to acrop plant. In certain embodiments, the crop plant is a food crop forhuman consumption, such as a fruit, vegetable, grain or tuber, such aspotatoes.

In other embodiments, the crop plant is a feed crop for producing e.g.,cereal grains (e.g., oats), corn, alfalfa, barley, and various kinds ofgrasses and hay.

In other embodiments, the crop plant is a fiber crop, such as cotton,flax, hemp, and bamboo.

In other embodiments, the crop plant is an oil crop for producing e.g.,corn, sunflower, canola, safflower, and olive oils.

In other embodiments, the crop plant is an ornamental crop, such asshade trees, flowering trees, shrubs, flowers, and grasses

In other embodiments, the crop plant is an industrial crop, such as aHevea tree for producing rubber.

Exemplary plants that can be used for prophylaxis or treatment withMoMo30 compositions include the class of higher and lower plants,including angiosperms (monocotyledonous and dicotyledonous plants),gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, andmulticellular algae. Preferably, plants for use in the methods of thepresent invention include any vascular plant, for example monocotyledonsor dicotyledons or gymnosperms, including, but not limited to alfalfa,apple, Arabidopsis, banana, barley, canola, castor bean, chrysanthemum,clover, cocoa, coffee, cotton, cottonseed, corn, crambe, cranberry,cucumber, dendrobium, dioscorea, eucalyptus, fescue, flax, gladiolus,liliacea, linseed, millet, muskmelon, mustard, oat, oil palm, oilseedrape, papaya, peanut, pineapple, ornamental plants, Phaseolus, potato,rapeseed, rice, rye, ryegrass, safflower, sesame, sorghum, soybean,sugarbeet, sugarcane, sunflower, strawberry, tobacco, tomato, turfgrass,wheat and vegetable crops such as lettuce, celery, broccoli,cauliflower, cucurbits; fruit and nut trees, such as apple, pear, peach,orange, grapefruit, lemon, lime, almond, pecan, walnut, hazel; vines,such as grapes, kiwi, hops; fruit shrubs and brambles, such asraspberry, blackberry, gooseberry; forest trees, such as ash, pine, fir,maple, oak, chestnut, popular; with alfalfa, canola, castor bean, corn,cotton, crambe, flax, linseed, mustard, oil palm, oilseed rape, peanut,potato, rice, safflower, sesame, soybean, sugarbeet, sunflower, tobacco,tomato, and wheat preferred. More preferably, plants for use in themethods of the present invention include any crop plant, for example,forage crop, oilseed crop, grain crop, fruit crop, vegetable crop, fibercrop, spice crop, nut crop, turf crop, sugar crop, beverage crop, andforest crop. In a highly preferred aspect, the crop plant used in amethod is a soybean plant. In another highly preferred aspect, the cropplant is wheat. In another highly preferred aspect, the crop plant iscorn. In another highly preferred aspect, the crop plant is cotton. Inanother highly preferred aspect, the crop plant is alfalfa. In anotherhighly preferred aspect, the crop plant is sugarbeet. In another highlypreferred aspect, the crop plant is rice. In another highly preferredaspect, the crop plant is potato. In another highly preferred aspect,the crop plant is tomato.

In some embodiments, the prevention and reduction of pathogen infectionsor related disease symptoms are reduced at least about 10% from a plantuntreated by the hevamine A-related composition. Preferably, theinfection, symptoms or both are prevented or reduced at least about 20%,30%, 40%, 50%, 60%, 70%, 80% compared to infection, symptoms or both ona plant not treated with a MoMo30 composition. A disease infection maybe measured by any reproducible means of measurement. In one aspect,infection may be measured by counting lesions or pustules visible to thenaked eye, or at a specified magnification. In a preferred aspect, thespecified magnification is 2×, 3×, 4×, 5×, 10×, or 50×.

In some embodiments, methods of preventing disease in a plant areprovided where only a portion of the plant is contacted with thehevamine A-related composition, yet untreated portions of the plant arealso protected from disease. In one aspect, only about 5%, 10%, 20%,30%, 50%, 75% or 90% of the plant is contacted with the hevamineA-related composition. The percentage of plant contacted by the hevamineA-related composition may be measured by any reproducible means ofmeasurement.

In some embodiments, the plant pathogen is a fungus. The method of thepresent invention can be used to control, prevent or treat infectionfrom a wide array of plant pathogens that include obligate parasites,facultative parasites, and facultative saprophytes, which include, butare not limited to the following: Ascomycete fungi such as of the generaVenturia, Podosphaera, Erysiphe, Monolinia, Mycosphaerella, andUncinula; Basidiomycete fungi such as from the genera Hemileia,Rhizoctonia, and Puccinia; Fungi imperfecti such as the genera Botrytis,Helminthosporium, Rhynchosporium, Fusarium (i.e., F. monoliforme),Septoria, Cercospora, Alternaria, Pyricularia, and Pseudocercosporella(i.e., P. herpotrichoides); Oomycete fungi such as from the generaPhytophthora (i.e., P. parasitica. P. medicaginis, P. megasperma),Peronospora (i.e, P. tabacina), Bremia, Pythium, and Plasmopara; as wellas other fungi such as Scleropthora macrospora, Sclerophthora rayissiae,Sclerospora graminicola, Peronosclerospora sorghi, Peronosclerosporaphihppinensis, Peronosclerospora sacchari and Peronosclerospora maydis,Physopella zeae, Cercospora zeaemaydis, Colletotrichum graminicola,Gibberella zeae, Exserohilum turcicum, Kabatiellu zeae, and Bipolarismaydis.

Exemplary fungal pathogens include e.g., genera Alternaria (e.g., A.solani); Fusarium (e.g., F. culmorum, F. graminearum, F. culmorum, F.graminearum, F. oxysporum, F. roseum); Microdochium (e.g. M. nivale);Magnaporthe; Monilia (e.g. M. fructigenae, M. laxa); Aspergillus (e.g.,A. paraciticus); Penicillium; Aspergillus; Austropuccinia; Hemileia(e.g., H. vastatrix); Botrytis (e.g. B. cinerea, B. fabae, B. ricini, B.elliptica); Cronartium; Eutypa; Phaeomoniella; Phaeoacremonium;Phomopsis; Cercospora; Mycosphaerella; Bipolaris; Sclerotinia; Pythium(e.g., P. aphanidermatum, P. irregulare, P. ultimum); Phytophthora (e.g.P. infestans, P. fragariae, P. cactorum, P. sojae, P. cinnamomi, P.citricola, P. citrophthora, P. cryptogea, P. drecshsleri, P. infestans,P. nicotianae); Rhizoctonia (e.g. R. solani, R. cerealis); Puccinia(rusts; e.g., P. striiformis, P. graminis tritici, P. recondite, P.hordei, P. triticina, P. triticiduri); Leveillula; Microsphaera (e.g. M.vaccinia); Podosphaera; Odium; Sphaerotheca; Peronospora; Cercospora;Erysiphe; Uncinula; Phomopsis (e.g. P. viticola, P. rachis, P.vaccinii); Monilinia (e.g. M. vaciniicormbosi, M. fructicola);Phragmidium (e.g. yellow rust); Drepanopeziza sp. (e.g. anthracnose);Erysiphe (e.g., E. graminis); Kuehneola (e.g. cane and leaf rust);Sphaerulina (e.g. orange rust); Arthuriomyces (e.g. powdery mildew);Mycosphaerella sp. (leaf spot; e.g. M. pinodes, M. graminicola);Dendrophoma (e.g., D. obscurans); Diplocarpon (e.g., D. earliana);Godronia (e.g., G. cassandrac); Exobasidium (e.g., E. vaccinii);Gymnosporangium (e.g., apple rust); Leucostoma (e.g., L. cincta or L.persoonii); Apiosporina (e.g., A. morbosa); Melampsora (e.g., M. lini);Sclerotium (e.g., S. rolfsii); Septoria sp. (e.g., S. tritici, S.nodorum, S. passerinii); Phaeosphaeria (e.g., P. nodorum); Tapesia(e.g., T. yallundae, T. acuiformis); Thielaviopsus (e.g., T. basicola);Gaeumannomyces (e.g., G. graminis); Erysiphe (e.g., E. graminis, E.cichoracearum, E. beticola); Drechslera (e.g., D. triticirepentis);Phakopsora (e.g., Phakopsora pachyrhizi, Phakopsora meibomiae,Phakopsora euvitis); Pyrenophora (e.g., P. teres); Cochliobolus (e.g.,C. sativus anamorphe: Bipolaris sorokiniana); Rhynchosporium (e.g., R.secalis); Ascochyta (e.g., A. pisi); Peronospora (e.g., P. pisi, P.manchurica); Rhizopus; Trichoderma; Magnaporthe (e.g., M. grisea, M.oryzae); Sphaerotheca (e.g., S. fuliginea and S. macularis); Leveillula(e.g., L. taurica); Cladosporium; Colletotrichum (e.g., C. acutatum);Venturia (V. inaequalis); Podosphaera (e.g., P. leucotricha); Uncinula(e.g., U. necator); Guignardia (e.g., G. bidwellii); Plasmopara (e.g.,P. viticola); Ramularia (e.g., R. beticola); Cercospora (e.g., C.beticola); Stagonospora (e.g., S. nodorum); Ustilago (e.g., U. maydis);Uromyces; Verticillium; Drechslera teres f. maculate; Ramularia collocygni; Ophiocladium horde; and Blumeria graminis.

Particularly preferred pathogens include, but are not limited to:Puccinia, Rhizoctonia, GGT, stripe rust, Asian soybean rust (Phakopsorapachyrhizi), Fusarium species, Verticillium species, gray leaf spot,Phytophthora species and corn rust.

Diseases controlled, prevented or treated with the MoMo30 compositionsof the present application include, for example, diseases of alfalfaplants such as root rot (Phytophora medicaginis, P. megasperma); riceplant such as rice blast (Pyricularia oryzae), Helminthosporium leafblight (Helminthosporium oryzae, Cochliobolus miyabeanus), Bakanaedisease (Gibberella fujikuroi), seedling blight (Rhizopus oryzae),sheath blight (Rhizoctonia solani), and so on, those of oat such ascrown rust (Puccinia coronata), and so on, those of barley such aspowdery mildew (Erysiphe graminis), scald (Rhynchsporium secalis),spot-blotch (Cochliobolus sativus), yellow mottleleaf (Helminthosporiumgramineum, Pyrenophora gramineum), net blotch (Pyrenophra teres),stinking smut (Tilletia caries), loose smut (Ustilago nuda), and so on,those of wheat such as powdery mildew (Erysiphe graminis), glume-blotch(Leptosphaeria nodorum, Septoria nodorum), stripe rust (Pucciniastriiformis), Typhula snow blight (Typhula incarnata), eye spot(Pseudocercosporella herpotrichoides), snow mold (Calonectriagraminicola, Fusarium nivale), stem rust (Puccinia graminis), black snowblight (Typhula ishikariensis), scab (Gibberella zeae), leaf rust(Puccinia recondita, Puccinia triticina), stripe (Helminthosporiumgramineum), stinking smut (Tilletia caries), speckled leaf blight(Septoria tritici), loose smut (Ustilago tritici), and so on, those ofcorn such as damping-off (Pythium debaryanum), and so on, those of ryesuch as purple snow mold (Fusarium nivale), and so on, those of potatosuch as late blight (Phytophthora infestans), and so on, those oftobacco plant such as downy mildew (Peronospora tabacina), foot rot(Phytophthora parasitica var), septoria blight (Cercospora nicotianae),and so on, those of sugar beet such as leaf spot (Cercospora beticola),damping-off (Pythium debaryanum, Rhizoctonia solani, Pythiumaphanidermatum), and so on, those of paprika such as gray mold (Botrytiscinerea), and so on, those of kidney bean such as gray mold (Botrytiscinerea), sclerotinia seed rot (sclerotial rot) (Sclerotiniasclerotiorum), southern blight (Corticium rolfsii), and so on, those ofbroad bean such as powdery mildew (Erysiphe polygoni, Sphaerothecafuliginea), rust (Uromyces fabae, Uromyces phaseoli), gray mold(Botrytis cinerea), and so on, those of peanut such as Ascochyta spot(Mycosphaerella arachidicola), and so on, those of cabbage such asdamping blight (Rhizoctonia solani), and so on, those of cucumber suchas powdery mildew (Sphaerotheca fuliginea), stem rot (Fusariumoxysporum), gummy stem blight (Mycosphaerella melonis), downy mildew(Pseudoperonospora cubensis), gray mold (Botrytis cinerea), sclerotialseed rot (Sclerotinia sclerotiorum), anthracnose (Colletotrichumlagenarium), damping blight (Fusarium oxysporum, Pythium aphanidermatum,Rhizoctonia solani), and so on, those of KOMATSUNA such as Alternariasooty spot (Alternaria brassicicola), club root (Plasmodiophorabrassicae), and so on, those of celery such as speckled leaf blotch(Septoria apii), and soon, those of radish such as yellows (Fusariumoxysporum), and so on, those of tomato such as Fusarium wilt (Fusariumoxysporum), foot rot (Phytophthora infestans), ring leaf-spot(Alternaria solani), gray mold (Botrytis cinerea), leaf blight(Phytophthora capsici), black rot (Alternaria tomato), and so on, thoseof eggplant such as brown rot (Phytophthora capsici), vascular wiltpathogens, e.g. Verticillium wilt (Verticillium alboatrum. V. dahliae),and so on, those of Chinese cabbage such as black rot (Alternariajaponica), club root (Plasmodiophora brassicae), and so on, those ofsweet pepper such as foot rot (Phytophthora capsici), gray mold(Botrytis cinerea), and so on, those of lettuce such as gray mold(Botrytis cinerea), and so on, those of citrus fruits such as pod andstem blight (Diaporthe citri), and so on, those of pear such as scab(Venturia nashicola), black rot (Alternaria kikuchiana), brown-spot(Gymnosporangium haraeanum), and so on, those of grape such as downymildew (Plasmopara viticola), gray mold (Botrytis cinerea), Sphacelomascab (Elsinoe ampelina), and so on, those of peach such as leaf curl(Taphrina deformans), shot hole (Mycosphaerella cerasella), and so on,those of apple such as powdery mildew (Podosphaera leucotria), scab(Cladsporium carpophilum), gray mold (Botrytis cinerea), black rot(Venturia inaegualis), brown spot (Gymnosporangium yamadae), white rootrot (Rosellinia nectrix), Alternaria leaf spot (Alternaria mali), and soon, and other diseases of grains, fruits and vegetables such as oil-seedrape, sunflower, carrot, pepper, strawberry, melon, kiwi fruit, onion,leek, sweet potato, fig, ume, asparagus, persimmon, soybean, adzukibean,watermelon, crown daisy, spinach, tea and so on. Additional fungalpathogens and plant diseases are described in U.S. Pat. No. 10,329,580,which is expressly incorporated herein by reference in its entirety.

In some embodiments, the plant pathogen is a bacterium. Bacteria cancause a number of diseases on ornamental and agronomic crops. Theseinclude leaf spotting on English ivy, fireblight on apples and pears,crown gall on stone fruits, and wilts in geraniums and cucurbits(cucumbers, squash and melons). Common plant pathogenic bacteriainclude, but are not limited to Erwinia spp., Dickeya spp., Pseudomonasspp., Xanthomonas spp., and Clavibacter spp. These pathogens may attackplant root systems, foliage, or a combination of both.

Exemplary Erwinia species include, but are not limited to E. amylovora,E. aphidicola, E. billingiae, E. mallotivora E. papayae, E. persicina,E. psidii, E. pyrifoliae, E. rhapontici, E. stewartia, E. toletana, andE. tracheiphila. Exemplary Dickeya species include, but are not limitedto D. chrysanthemi, D. dadantii, D. solani. Exemplary Pseudomonasspecies include, but are not limited to P. amygdale, P. avellanae, P.caricapapayae, P. cichorii, P. coronafaciens, P. ficuserectae, P.helianthi, P. meliae, P. savastanoi, P. syringae, P. tabaci, P. tomato,P. viridiflava. Exemplary Xanthomonas species include, but are notlimited to pathovars of X. albilineans, X. alfalfae, X. ampelina, X.arboricola, X. axonopodis, X. boreopolis, X. badrii, X. bromi, X.campestris, X. cassayae, X. citri, X. codiaei, X. cucurbitae, X.cyanopsidis, X. cynarae, X. euvesicatoria, X. fragariae, X. gardneri, X.holcicola, X. hortorum, X. hyacinthi, X. malvacearum, X. maltophilia, X.manihotis, X. melonis, X. oryzae, X. papavericola, X. perforans, X.phaseoli, X. pisi, X. populi, X. sacchari, X. theicola, X. translucens,X. vasicola, X. vesicatoria. Exemplary Clavibacter species include, butare not limited to Clavibacter michiganensis and Clavibactermichiganensis subsp. insidiosus.

Additional bacterial pathogens include mycoplasma and mycoplasma-like(phytoplasma) bacteria, such as Xylella fastidiosa, which causesPierce's disease and Phony Peach diseases, several Phytoplasma causingAster Yellows disease, Peach X disease, and Peach Yellow disease; S.kunkelii, causing corn stunt disease), as well as various rickettsia andrickettsia-like bacteria.

In some embodiments, the plant pathogen is a plant virus. In some cases,the plant virus is Tobacco mosaic virus, Tomato spotted wilt virus,Tomato yellow leaf curl virus, Cucumber mosaic virus, Potato virus Y,Cauliflower mosaic virus, African cassava mosaic virus, Plum pox virus,Brome mosaic virus, Potato virus X, Citrus tristeza virus, Barley yellowdwarf virus, Alfalfa dwarf virus Potato leafroll virus, or Tomato bushystunt virus.

III. Genetically Modified Plants

In another aspect, the present application provides a transgenic plantexpressing the MoMo30 protein of the present application. In oneembodiment, the present disclosure relates to a transgenic plant, plantpart, or plant cell, wherein the transgene comprises a polynucleotideencoding an amino acid sequence at least 95% identical to SEQ ID NO: 4and exhibits resistance or tolerance to a plant pathogen.

As used herein, the term “transgenic plant” refers to a plant thatcontains genetic material not found (i.e. “exogenous”) in a wild-typeplant of the same species, variety or cultivar. In accordance with thepresent application, the genetic material comprises a MoMo30 expressioncassette introduced into the plant by human manipulation. The transgenicplant may additionally contain sequences which are native to the plantbeing transformed, but wherein the “exogenous” gene has been altered inorder to alter the level or pattern of expression of the gene, forexample, by use of one or more heterologous regulatory elements and thelike.

As further described below, the expression cassette typically comprisesa polypeptide-encoding sequence operably linked (i.e., under regulatorycontrol of) to appropriate inducible or constitutive regulatorysequences that allow for the expression of the polypeptide. Theexpression cassette can be introduced into a plant by transformation orby breeding after transformation of a parent plant.

The plant or plant part for use in the present invention include plantsof any stage of plant development. Exemplary plant parts include leaves,roots, stems, fruit, seeds, tubers, bulbs, seeds, pollen, ovules,flowers, pods, stems, shoots, and combinations thereof. Preferably, theapplication occurs during the stages of germination, seedling growth,vegetative growth, and reproductive growth. More preferably,applications of the present invention occur during vegetative andreproductive growth stages. The stages of vegetative and reproductivegrowth are also referred to herein as “adult” or “mature” plants.

Methods of producing transgenic plants or transgenic plant cells arewell known to those of ordinary skill in the art. A transgenic plantcell or transgenic plant is obtained by either transforming anon-transgenic plant cell or plant to create the transgenic plant cellor plant, or planting transgenic plant seed to produce the transgenicplant cell or plant. Such a transgenic plant seed may be from an ROtransgenic plant or may be from a progeny of any generation thereof thatinherits a given transgenic sequence from a starting transgenic parentplant. As used herein, the term “RO transgenic plant” refers to a plantthat has been genetically transformed or has been regenerated from aplant cell or cells that have been genetically transformed.

Transgenic plants can now be produced by a variety of differenttransformation methods including, but not limited to, electroporation;microinjection; microprojectile bombardment, also known as particleacceleration or biolistic bombardment; viral-mediated transformation;and Agrobacterium-mediated transformation. See, for example, U.S. Pat.Nos. 5,405,765; 5,472,869; 5,538,877; 5,538,880; 5,550,318; 5,641,664;5,736,369, 8,937,214, 10,329,580, and U.S. Patent Publication No.2022/0135997; each of which is expressly incorporated herein byreference in their entirety.

In one aspect, the present application provides a transgenic plant,transgenic plant part, or transgenic plant cell, comprising: a stablyintegrated DNA expression construct comprising a polynucleotidecomprising a polynucleotide containing a nucleotide sequence at least95% identical to SEQ ID NO: 1 or SEQ ID NO: 2, or a polynucleotideencoding a protein comprising an amino acid sequence at least 95%identical to SEQ ID NO: 3 or SEQ ID NO: 4, wherein the transgenic plantexhibits increased resistance to at least one bacterial, fungal, orviral infection as compared to a control plant lacking the recombinantDNA expression construct under the same condition.

The MoMo30 transgene may be introduced in any plant susceptible to aplant pathogen, including any of the plants described above. In certainpreferred embodiments, the transgenic plant is a crop plant. Exemplarycrop plants include, but are not limited to wheat, corn, rice, barley,cotton, canola, alfalfa, sugarbeet, potato and tomato.

The MoMo30 transgene may be introduced in any plant susceptible to aplant pathogen, including any of the plants described above. In certainpreferred embodiments, the transgenic plant is a crop plant. Exemplarycrop plants include, but are not limited to a forage crop, oilseed crop,grain crop, fruit crop, vegetable crop, fiber crop, spice crop, nutcrop, turf crop, sugar crop, beverage crop, and forest crop. In certainpreferred embodiments, crop plant is wheat, corn, rice, barley, cotton,canola, alfalfa, sugarbeet, potato and tomato.

Preferred components likely to be included with vectors used in thepresent application are as follows.

i. Regulatory Elements

Exemplary promoters for expression of a transgene include plantpromoters, such as the Cauliflower Mosaic Virus (CaMV) 35S promoter, orother promoters, such as CaMV 19S, nopaline synthase (Nos), alcoholdehydrogenase (Adh), sucrose synthase, α-tubulin, actin, chlorophylla/b-binding protein (Cab), phosphoenolpyruvate carboxylase (PEPCKase) orthose associated with the R gene complex. Tissue specific promoters suchas root cell promoters and tissue specific enhancers are alsocontemplated to be particularly useful, as are inducible promoters suchas ABA- and turgor-inducible promoters. In certain aspects, a promoterfor use according to the application is an ePCISV, TubA, eFMV, FMV,e35S, 35S or Ract1 promoter.

In certain aspects, transformation events comprised in transgenic plantsaccording to the application comprise a plurality of promoter sequences.In certain aspects, a promoter sequence is repeated no more than about2, 3, 4, or 5 times in a single transformation event. In otherembodiments, identical or highly homologous promoter sequences arelinked to at least 2, 3, 4, 5 or more transgenes in a singletransformation event. In certain embodiments, a transformation eventcomprising a plurality of transgenes comprises at least 2, 3, 4, 5, 6,7, 8, 9 or 10 different promoter sequences.

In further embodiments, identical or highly homologous promotersequences are linked to transgenes that confer similar traits (e.g.,transgenes that confer insect resistance). In certain aspects, two ormore identical or highly homologous promoter sequences are separated byat least 1, 2 or 3 expression cassettes within a single transformationevent. In other embodiments, identical or highly homologous promotersequences are linked to two or more contiguous expression cassettes in asingle transformation event.

The DNA sequence between the transcription initiation site and the startof the coding sequence, i.e., the untranslated leader sequence, can alsoinfluence gene expression. One may thus wish to employ a particularleader sequence with a transformation construct of the application.Preferred leader sequences are contemplated to include those whichcomprise sequences predicted to direct optimum expression of theattached gene, i.e., to include a preferred consensus leader sequencewhich may increase or maintain mRNA stability and prevent inappropriateinitiation of translation. The choice of such sequences will be known tothose of skill in the art in light of the present disclosure. Sequencesthat are derived from genes that are highly expressed in plants willtypically be preferred.

In another aspect, the present application provides a method forproducing foregoing transgenic plant, comprising the steps of: (a)stably transforming a host plant or plant cell with a polynucleotidecomprising a polynucleotide containing a nucleotide sequence at least95% identical to SEQ ID NO: 1 or SEQ ID NO: 2, or a polynucleotideencoding a protein comprising an amino acid sequence at least 95%identical to SEQ ID NO: 3 or SEQ ID NO: 4; and (b) producing thetransgenic plant or plant cell, where the transgenic plant or transgenicplant cell is identified that expresses the expression construct in anamount sufficient to provide increased resistance to at least onebacterial or fungal infection as compared to a control plant lacking therecombinant DNA expression construct under the same condition.

A. Plant Transformation and Transgene Expression Constructs

Certain embodiments of the present application relate to theconstruction and use of plant transformation constructs. Generally, theMoMo30 coding sequences are provided operably linked to a promoter(e.g., a heterologous promoter). Expression constructs are also providedcomprising these sequences, as are plants and plant cells transformedwith the sequences.

The construction of vectors which may be employed in conjunction withplant transformation techniques using these or other sequences accordingto the application will be known to those of skill of the art in lightof the present disclosure. The techniques of the present application arethus not limited to any particular nucleic acid sequences.

Particularly useful for transformation are expression cassettes whichhave been isolated from such vectors. DNA segments used for transformingplant cells will, of course, generally comprise the RNA coding sequence,cDNA, gene or genes which one desires to introduce into and haveexpressed in the host cells. These DNA segments can further includestructures such as promoters, enhancers, polylinkers, or even regulatorygenes as desired. The DNA segment or gene chosen for cellularintroduction will often encode a protein which will be expressed in theresultant recombinant cells resulting in a screenable or selectabletrait and/or which will impart an improved phenotype to the resultingtransgenic plant. However, this may not always be the case, and thepresent application also encompasses transgenic plants incorporatingnon-expressed transgenes. Preferred components likely to be includedwith vectors used in the present application are as follows.

i. Regulatory Elements

Exemplary promoters for expression of a transgene include plantpromoters, such as the Cauliflower Mosaic Virus (CaMV) 35S promoter, orother promoters, such as CaMV 19S, nopaline synthase (Nos), alcoholdehydrogenase (Adh), sucrose synthase, α-tubulin, actin, chlorophylla/b-binding protein (Cab), phosphoenolpyruvate carboxylase (PEPCKase) orthose associated with the R gene complex. Tissue specific promoters suchas root cell promoters and tissue specific enhancers are alsocontemplated to be particularly useful, as are inducible promoters suchas ABA- and turgor-inducible promoters. In certain aspects, a promoterfor use according to the application is an ePCISV, TubA, eFMV, FMV,e35S, 35S or Ract1 promoter.

In certain aspects, transformation events comprised in transgenic plantsaccording to the application comprise a plurality of promoter sequences.In certain aspects, a promoter sequence is repeated no more than about2, 3, 4, or 5 times in a single transformation event. In otherembodiments, identical or highly homologous promoter sequences arelinked to at least 2, 3, 4, 5 or more transgenes in a singletransformation event. In certain embodiments, a transformation eventcomprising a plurality of transgenes comprises at least 2, 3, 4, 5, 6,7, 8, 9 or 10 different promoter sequences.

In further embodiments, identical or highly homologous promotersequences are linked to transgenes that confer similar traits (e.g.,transgenes that confer insect resistance). In certain aspects, two ormore identical or highly homologous promoter sequences are separated byat least 1, 2 or 3 expression cassettes within a single transformationevent. In other embodiments, identical or highly homologous promotersequences are linked to two or more contiguous expression cassettes in asingle transformation event.

The DNA sequence between the transcription initiation site and the startof the coding sequence, i.e., the untranslated leader sequence, can alsoinfluence gene expression. One may thus wish to employ a particularleader sequence with a transformation construct of the application.Preferred leader sequences are contemplated to include those whichcomprise sequences predicted to direct optimum expression of theattached gene, i.e., to include a preferred consensus leader sequencewhich may increase or maintain mRNA stability and prevent inappropriateinitiation of translation. The choice of such sequences will be known tothose of skill in the art in light of the present disclosure. Sequencesthat are derived from genes that are highly expressed in plants willtypically be preferred.

It is specifically envisioned that transgene coding sequences may beintroduced under the control of novel promoters or enhancers, etc., orhomologous or tissue specific promoters or control elements. Vectors foruse in tissue-specific targeting of genes in transgenic plants willtypically include tissue-specific promoters and may also include othertissue-specific control elements such as enhancer sequences. Promotersthat direct specific or enhanced expression in certain plant tissues areknown to those of skill in the art in light of the present disclosure.These include, for example, the rbcS promoter, specific for greentissue; the ocs, nos and mas promoters which have higher activity inroots or wounded leaf tissue; a truncated (−90 to +8) 35S promoter whichdirects enhanced expression in roots, and an α-tubulin gene that alsodirects expression in roots.

ii. Terminators

Transformation constructs prepared in accordance with the applicationwill typically include a 3′ end DNA sequence that acts as a signal toterminate transcription and allow for the poly-adenylation of the mRNAproduced by coding sequences operably linked to a transgene. Terminatorswhich are deemed to be particularly useful in this context include thosefrom the nopaline synthase gene of Agrobacterium tumefaciens (nos 3′end), the terminator for the T7 transcript from the octopine synthasegene of Agrobacterium tumefaciens, and the 3′ end of the proteaseinhibitor I or II genes from potato or tomato. Regulatory elements suchas an Adh intron, sucrose synthase intron or TMV omega element, mayfurther be included where desired. In certain aspects, a terminator foruse according to the application is a Hsp17, TubA, Ara5, 35S, nos or Tr7terminator.

In certain aspects, transformation events comprised in transgenic plantsaccording to the application comprise a plurality of terminatorsequences. In certain aspects, a terminator sequence is repeated no morethan about 2, 3, 4, or 5 times in a single transformation event. Inother embodiments, identical or highly homologous terminator sequencesare linked to at least 2, 3, 4, 5 or more transgenes in a singletransformation event. In certain embodiments, a transformation eventcomprising a plurality of transgenes comprises at least 2, 3, 4, 5, 6,7, 8, 9 or 10 different terminator sequences. In further embodiments,identical or highly homologous terminator sequences are linked totransgenes that confer similar traits (e.g., transgenes that conferinsect resistance). In certain aspects, two or more identical or highlyhomologous terminator sequences are separated by at least 1, 2 or 3expression cassettes with a single transformation event. In otherembodiments, identical or highly homologous terminator sequences arelinked to two or more contiguous expression cassettes in a singletransformation event.

iii. Intron Sequences

In certain aspects, intron sequences are included an expression cassetteand may enhance transgene expression. In certain aspects, an intron foruse according to the application is a Ract1, TubA, Sus1 or Hsp70 intron.

In certain aspects, transformation events comprised in transgenic plantsaccording to the application comprise a plurality of intron sequences.In certain aspects, an intron sequence is repeated no more than about 2,3, 4, or 5 times in a single transformation event. In other embodiments,identical or highly homologous intron sequences are linked to at least2, 3, 4, 5 or more transgenes in a single transformation event. Incertain embodiments, a transformation event comprising a plurality oftransgenes comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 differentintron sequences.

In further embodiments, identical or highly homologous intron sequencesare linked to transgenes that confer similar traits (e.g., transgenesthat confer insect resistance). In certain aspects, two or moreidentical or highly homologous intron sequences are separated by atleast 1, 2 or 3 expression cassettes within a single transformationevent. In other embodiments, identical or highly homologous intronsequences are linked to two or more contiguous expression cassettes in asingle transformation event.

iv. Transit or Signal Peptides

Sequences that are joined to the coding sequence of an expressed gene,which are removed post-translationally from the initial translationproduct and which facilitate the transport of the protein into orthrough intracellular or extracellular membranes, are termed transit(usually into vacuoles, vesicles, plastids and other intracellularorganelles) and signal sequences (usually to the endoplasmic reticulum,golgi apparatus and outside of the cellular membrane). By facilitatingthe transport of the protein into compartments inside and outside thecell, these sequences may increase the accumulation of gene productprotecting them from proteolytic degradation. These sequences also allowfor additional mRNA sequences from highly expressed genes to be attachedto the coding sequence of the genes. Since mRNA being translated byribosomes is more stable than naked mRNA, the presence of translatablemRNA in front of the gene may increase the overall stability of the mRNAtranscript from the gene and thereby increase synthesis of the geneproduct. Since transit and signal sequences are usuallypost-translationally removed from the initial translation product, theuse of these sequences allows for the addition of extra translatedsequences that may not appear on the final polypeptide. It further iscontemplated that targeting of certain proteins may be desirable inorder to enhance the stability of the protein (U.S. Pat. No. 5,545,818,incorporated herein by reference in its entirety).

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This generally will be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit, or signal, peptide willtransport the protein to a particular intracellular, or extracellulardestination, respectively, and will then be post-translationallyremoved.

v. Marker Genes

By employing a selectable or screenable marker protein, one can provideor enhance the ability to identify transformants. “Marker genes” aregenes that impart a distinct phenotype to cells expressing the markerprotein and thus allow such transformed cells to be distinguished fromcells that do not have the marker. Such genes may encode either aselectable or screenable marker, depending on whether the marker confersa trait which one can “select” for by chemical means, i.e., through theuse of a selective agent (e.g., a herbicide, antibiotic, or the like),or whether it is simply a trait that one can identify throughobservation or testing, i.e., by “screening” (e.g., the greenfluorescent protein). Of course, many examples of suitable markerproteins are known to the art and can be employed in the practice of theapplication.

Included within the terms “selectable” or “screenable markers” also aregenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which are secretable antigens that can be identified byantibody interaction, or even secretable enzymes which can be detectedby their catalytic activity. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA; small active enzymes detectable in extracellular solution (e.g.,α-amylase, β-lactamase, phosphinothricin acetyltransferase); andproteins that are inserted or trapped in the cell wall (e.g., proteinsthat include a leader sequence such as that found in the expression unitof extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a protein that becomes sequestered in the cell wall, and whichprotein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

Many selectable marker coding regions are known and could be used withthe present application including, but not limited to, neo, whichprovides kanamycin resistance and can be selected for using kanamycin,G418, paromomycin, etc.; bar, which confers bialaphos orphosphinothricin resistance; a mutant EPSP synthase protein conferringglyphosate resistance; a nitrilase such as bxn from Klebsiella ozaenaewhich confers resistance to bromoxynil; a mutant acetolactate synthase(ALS) which confers resistance to imidazolinone, sulfonylurea or otherALS inhibiting chemicals; a methotrexate resistant DHFR, a dalapondehalogenase that confers resistance to the herbicide dalapon; or amutated anthranilate synthase that confers resistance to 5-methyltryptophan.

An illustrative embodiment of selectable marker capable of being used insystems to select transformants are those that encode the enzymephosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes. The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, causing rapidaccumulation of ammonia and cell death.

Screenable markers that may be employed include a β-glucuronidase (GUS)or uidA gene which encodes an enzyme for which various chromogenicsubstrates are known; an R-locus gene, which encodes a product thatregulates the production of anthocyanin pigments (red color) in planttissues; a β-lactamase gene, which encodes an enzyme for which variouschromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a xylE gene, which encodes a catechol dioxygenase thatcan convert chromogenic catechols; an α-amylase gene; a tyrosinase gene,which encodes an enzyme capable of oxidizing tyrosine to DOPA anddopaquinone which in turn condenses to form the easily-detectablecompound melanin; a β-galactosidase gene, which encodes an enzyme forwhich there are chromogenic substrates; a green fluorescent protein, aluciferase gene, which allows for bioluminescence detection; an aequoringene, which may be employed in calcium-sensitive bioluminescencedetection.

Another screenable marker contemplated for use in the presentapplication is the firefly luciferase gene (lux), which allows forbioluminescence detection. The presence of the lux gene in transformedcells may be detected using, for example, X-ray film, scintillationcounting, fluorescent spectrophotometry, low-light video cameras, photoncounting cameras or multiwell luminometry. It also is envisioned thatthis system may be developed for population screening forbioluminescence, such as on tissue culture plates, or even for wholeplant screening. The gene which encodes green fluorescent protein (GFP)is also contemplated as a particularly useful reporter gene. Expressionof green fluorescent protein may be visualized in a cell or plant asfluorescence following illumination by particular wavelengths of light.

B. Tissue Cultures

Tissue cultures may be used in certain transformation techniques for thepreparation of cells for transformation and for the regeneration ofplants therefrom. As used herein, the term “regeneration” refers theprocess of growing a plant from a plant cell (e.g., plant protoplast,callus or explant). Maintenance of tissue cultures requires use of mediaand controlled environments. “Media” refers to the numerous nutrientmixtures that are used to grow cells in vitro, that is, outside of theintact living organism. The medium usually is a suspension of variouscategories of ingredients (salts, amino acids, growth regulators,sugars, buffers) that are required for growth of most cell types.However, each specific cell type requires a specific range of ingredientproportions for growth, and an even more specific range of formulas foroptimum growth. Rate of cell growth also will vary among culturesinitiated with the array of media that permit growth of that cell type.

Nutrient media is prepared as a liquid, but this may be solidified byadding the liquid to materials capable of providing a solid support.Agar is most commonly used for this purpose. Bacto™ agar (Difco-BD,Franklin Lakes, N.J.), Hazleton agar (Hazleton, Lenexa, Kans., USA),Gelrite® (Sigma, St. Louis, Mo.), PHYTAGEL (Sigma-Aldrich, St. Louis,Mo.), and GELGRO (ICN-MP Biochemicals, Irvine, Calif., USA) are specifictypes of solid support that are suitable for growth of plant cells intissue culture.

Some cell types will grow and divide either in liquid suspension or onsolid media. As disclosed herein, plant cells will grow in suspension oron solid medium, but regeneration of plants from suspension culturestypically requires transfer from liquid to solid media at some point indevelopment. The type and extent of differentiation of cells in culturewill be affected not only by the type of media used and by theenvironment, for example, pH, but also by whether media is solid orliquid.

Tissue that can be grown in a culture includes meristem cells, callus,immature embryos, hairy root cultures, and gametic cells such asmicrospores, pollen, sperm and egg cells. Callus may be initiated fromtissue sources including, but not limited to, immature embryos, seedlingapical meristems, root, leaf, microspores and the like. Those cellswhich are capable of proliferating as callus also are candidaterecipient cells for genetic transformation.

Somatic cells are of various types. Embryogenic cells are one example ofsomatic cells which may be induced to regenerate a plant through embryoformation. Non-embryogenic cells are those which typically will notrespond in such a fashion. Certain techniques may be used that enrichrecipient cells within a cell population, for example by manualselection and culture of friable, embryogenic tissue. Manual selectiontechniques which can be employed to select target cells may include,e.g., assessing cell morphology and differentiation, or may use variousphysical or biological means. Cryopreservation also is a possible methodof selecting for recipient cells.

Where employed, cultured cells may be grown either on solid supports orin the form of liquid suspensions. In either instance, nutrients may beprovided to the cells in the form of media, and environmental conditionscontrolled. There are many types of tissue culture media comprised ofvarious amino acids, salts, sugars, growth regulators and vitamins. Mostof the media employed in the practice of the application will have somesimilar components, but may differ in the composition and proportions oftheir ingredients depending on the particular application envisioned.For example, various cell types usually grow in more than one type ofmedia, but will exhibit different growth rates and differentmorphologies, depending on the growth media. In some media, cellssurvive but do not divide. Various types of media suitable for cultureof plant cells previously have been described. Examples of these mediainclude, but are not limited to, the N6 medium and MS media.

C. Methods for Genetic Transformation

Suitable methods for transformation of plant or other cells for use withthe present application are believed to include virtually any method bywhich DNA can be introduced into a cell, such as by direct delivery ofDNA such as by PEG-mediated transformation of protoplasts, bydesiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by Agrobacterium-mediatedtransformation, etc. Through the application of techniques such asthese, the cells of virtually any plant species may be stablytransformed, and these cells developed into transgenic plants.

i. Agrobacterium Mediated Transformation

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, U.S. Pat. No. 5,563,055.

Agrobacterium-mediated transformation is most efficient indicotyledonous plants and is the preferable method for transformation ofdicots, including Arabidopsis, tobacco, tomato, alfalfa and potato.Indeed, while Agrobacterium-mediated transformation has been routinelyused with dicotyledonous plants for a number of years, it has onlyrecently become applicable to monocotyledonous plants. Advances inAgrobacterium-mediated transformation techniques have now made thetechnique applicable to nearly all monocotyledonous plants. For example,Agrobacterium-mediated transformation techniques have now been appliedto e.g., rice, wheat, barley, alfalfa, and maize, among others.

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described. Moreover, recent technological advances invectors for Agrobacterium-mediated gene transfer have improved thearrangement of genes and restriction sites in the vectors to facilitatethe construction of vectors capable of expressing various polypeptidecoding genes. The vectors described have convenient multi-linker regionsflanked by a promoter and a polyadenylation site for direct expressionof inserted polypeptide coding genes and are suitable for presentpurposes. In addition, Agrobacterium containing both armed and disarmedTi genes can be used for the transformations. In those plant strainswhere Agrobacterium-mediated transformation is efficient, it is themethod of choice because of the facile and defined nature of the genetransfer.

ii. Electroporation

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wounding in acontrolled manner. Examples of some species which have been transformedby electroporation of intact cells include maize, wheat, tomato,soybean, and tobacco.

In some embodiments, protoplasts are employed for electroporationtransformation of plants. For example, the generation of transgenicsoybean plants by electroporation of cotyledon-derived protoplasts.Other examples of species for which protoplast transformation has beendescribed include barley, sorghum, maize, wheat, and tomato.

iii. Microprojectile Bombardment

Another method for delivering transforming DNA segments to plant cellsin accordance with the application is microprojectile bombardment. Inthis method, particles may be coated with nucleic acids and deliveredinto cells by a propelling force. Exemplary particles include thosecomprised of tungsten, platinum, and preferably, gold. It iscontemplated that in some instances DNA precipitation onto metalparticles would not be necessary for DNA delivery to a recipient cellusing microprojectile bombardment. However, it is contemplated thatparticles may contain DNA rather than be coated with DNA. Hence, it isproposed that DNA-coated particles may increase the level of DNAdelivery via particle bombardment but are not, in and of themselves,necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics® Particle Delivery System(Dupont), which can be used to propel particles coated with DNA or cellsthrough a screen, such as a stainless steel or nylon screen (e.g., NYTEXscreen; Sefar America, Depew, N.Y. USA), onto a filter surface coveredwith plant cells cultured in suspension. The screen disperses theparticles so that they are not delivered to the recipient cells in largeaggregates. Microprojectile bombardment techniques are widely applicableand may be used to transform virtually any plant species. Examples ofspecies for which have been transformed by microprojectile bombardmentinclude monocot species such as maize, barley, wheat, and sorghum; aswell as a number of dicots including tobacco, soybean, sunflower,peanut, cotton, tomato, and legumes in general.

iv. Other Transformation Methods

Transformation of protoplasts can be achieved using methods based oncalcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments.

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of plants from protoplasts arewell known in the art. Examples of the use of direct uptaketransformation of protoplasts include transformation of rice, sorghum,barley, oat, and maize (.

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as known in the art. Also, siliconcarbide fiber-mediated transformation may be used with or withoutprotoplasting. Transformation with this technique is accomplished byagitating silicon carbide fibers together with cells in a DNA solution.DNA passively enters as the cells are punctured.

C. Production and Characterization of Stably Transformed Plants

After effecting delivery of exogenous DNA to recipient cells, the nextsteps generally concern identifying the transformed cells for furtherculturing and plant regeneration. In order to improve the ability toidentify transformants, one may desire to employ a selectable orscreenable marker gene with a transformation vector prepared inaccordance with the application. In this case, one would then generallyassay the potentially transformed cell population by exposing the cellsto a selective agent or agents, or one would screen the cells for thedesired marker gene trait.

i. Selection

It is believed that DNA is introduced into only a small percentage oftarget cells in any one experiment. In order to provide an efficientsystem for identification of those cells receiving DNA and integratingit into their genomes one may employ a means for selecting those cellsthat are stably transformed. One exemplary embodiment of such a methodis to introduce into the host cell, a marker gene which confersresistance to some normally inhibitory agent, such as an antibiotic orherbicide. Examples of antibiotics which may be used include theaminoglycoside antibiotics neomycin, kanamycin and paromomycin, or theantibiotic hygromycin. Resistance to the aminoglycoside antibiotics isconferred by aminoglycoside phosphotransferase enzymes such as neomycinphosphotransferase II (NPT II) or NPT I, whereas resistance tohygromycin is conferred by hygromycin phosphotransferase.

Potentially transformed cells then are exposed to the selective agent.In the population of surviving cells will be those cells where,generally, the resistance-conferring gene has been integrated andexpressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of the exogenous DNA.

One herbicide which constitutes a desirable selection agent is the broadspectrum herbicide bialaphos. Bialaphos is a tripeptide antibioticproduced by Streptomyces hygroscopicus and is composed ofphosphinothricin (PPT), an analogue of L-glutamic acid, and twoL-alanine residues. Upon removal of the L-alanine residues byintracellular peptidases, the PPT is released and is a potent inhibitorof glutamine synthetase (GS), a pivotal enzyme involved in ammoniaassimilation and nitrogen metabolism. Synthetic PPT, the activeingredient in the herbicide Liberty™ also is effective as a selectionagent. Inhibition of GS in plants by PPT causes the rapid accumulationof ammonia and death of the plant cells.

The organism producing bialaphos and other species of the genusStreptomyces also synthesizes an enzyme phosphinothricin acetyltransferase (PAT) which is encoded by the bar gene in Streptomyceshygroscopicus and the pat gene in Streptomyces viridochromogenes. Theuse of the herbicide tolerance gene encoding phosphinothricin acetyltransferase (PAT) is referred to in DE 3642 829 A, wherein the gene isisolated from Streptomyces viridochromogenes. In the bacterial sourceorganism, this enzyme acetylates the free amino group of PPT preventingauto-toxicity. The bar gene has been cloned and expressed in transgenictobacco, tomato, potato, Brassica, and maize (U.S. Pat. No. 5,550,318).In previous reports, some transgenic plants which expressed theresistance gene were completely resistant to commercial formulations ofPPT and bialaphos in greenhouses.

Another example of a herbicide which is useful for selection oftransformed cell lines in the practice of the application is the broadspectrum herbicide glyphosate. Glyphosate inhibits the action of theenzyme EPSPS which is active in the aromatic amino acid biosyntheticpathway. Inhibition of this enzyme leads to starvation for the aminoacids phenylalanine, tyrosine, and tryptophan and secondary metabolitesderived thereof. U.S. Pat. No. 4,535,060 describes the isolation ofEPSPS mutations which confer glyphosate resistance on the Salmonellatyphimurium gene for EPSPS, aroA. The EPSPS gene was cloned from Zeamays and mutations similar to those found in a glyphosate resistant aroAgene were introduced in vitro. Mutant genes encoding glyphosateresistant EPSPS enzymes are described in, for example, U.S. Pat. No.6,566,587. The best characterized mutant EPSPS gene conferringglyphosate resistance comprises amino acid changes at residues 102 and106, although it is anticipated that other mutations will also be useful(U.S. Pat. No. 6,566,587).

To use the bar-bialaphos or the EPSPS-glyphosate selective system,transformed tissue is cultured for 0-28 days on nonselective medium andsubsequently transferred to medium containing from 1-3 mg/l bialaphos or1-3 mM glyphosate as appropriate. While ranges of 1-3 mg/l bialaphos or1-3 mM glyphosate will typically be preferred, it is proposed thatranges of 0.1-50 mg/l bialaphos or 0.1-50 mM glyphosate will findutility.

It further is contemplated that the herbicide DALAPON,2,2-dichloropropionic acid, may be useful for identification oftransformed cells. The enzyme 2,2-dichloropropionic acid dehalogenase(deh) inactivates the herbicidal activity of 2,2-dichloropropionic acidand therefore confers herbicidal resistance on cells or plantsexpressing a gene encoding the dehalogenase enzyme (U.S. Pat. No.5,508,468).

Alternatively, a gene encoding anthranilate synthase, which confersresistance to certain amino acid analogs, e.g., 5-methyltryptophan or6-methyl anthranilate, may be useful as a selectable marker gene. Theuse of an anthranilate synthase gene as a selectable marker wasdescribed in U.S. Pat. No. 5,508,468.

An example of a screenable marker trait is the enzyme luciferase. In thepresence of the substrate luciferin, cells expressing luciferase emitlight which can be detected on photographic or x-ray film, in aluminometer (or liquid scintillation counter), by devices that enhancenight vision, or by a highly light sensitive video camera, such as aphoton counting camera. These assays are nondestructive and transformedcells may be cultured further following identification. The photoncounting camera is especially valuable as it allows one to identifyspecific cells or groups of cells which are expressing luciferase andmanipulate those in real time. Another screenable marker which may beused in a similar fashion is the gene coding for green fluorescentprotein.

It further is contemplated that combinations of screenable andselectable markers will be useful for identification of transformedcells. In some cell or tissue types a selection agent, such as bialaphosor glyphosate, may either not provide enough killing activity to clearlyrecognize transformed cells or may cause substantial nonselectiveinhibition of transformants and nontransformants alike, thus causing theselection technique to not be effective. It is proposed that selectionwith a growth inhibiting compound, such as bialaphos or glyphosate atconcentrations below those that cause 100% inhibition followed byscreening of growing tissue for expression of a screenable marker genesuch as luciferase would allow one to recover transformants from cell ortissue types that are not amenable to selection alone. It is proposedthat combinations of selection and screening may enable one to identifytransformants in a wider variety of cell and tissue types. This may beefficiently achieved using a gene fusion between a selectable markergene and a screenable marker gene, for example, between an NPTII geneand a GFP gene.

ii. Regeneration and Seed Production

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, may be cultured in mediathat supports regeneration of plants. In an exemplary embodiment, MS andN6 media may be modified by including further substances such as growthregulators. One such growth regulator is dicamba or 2,4-D. However,other growth regulators may be employed, including NAA, NAA+2,4-D orpicloram. Media improvement in these and like ways has been found tofacilitate the growth of cells at specific developmental stages. Tissuemay be maintained on a basic media with growth regulators untilsufficient tissue is available to begin plant regeneration efforts, orfollowing repeated rounds of manual selection, until the morphology ofthe tissue is suitable for regeneration, at least 2 wk, then transferredto media conducive to maturation of embryoids. Cultures are transferredevery 2 wk on this medium. Shoot development will signal the time totransfer to medium lacking growth regulators.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, will then beallowed to mature into plants. Developing plantlets are transferred tosoiless plant growth mix, and hardened, e.g., in an environmentallycontrolled chamber, for example, at about 85% relative humidity, 600 ppmCO2, and 25-250 microeinsteins m⁻²s⁻¹ of light. Plants are preferablymatured either in a growth chamber or greenhouse. Plants can beregenerated from about 6 wk to 10 months after a transformant isidentified, depending on the initial tissue. During regeneration, cellsare grown on solid media in tissue culture vessels. Illustrativeembodiments of such vessels are petri dishes and Plantcon™ containers(MP-ICN Biomedicals, Solon, Ohio, USA). Regenerating plants arepreferably grown at about 19 to 28° C. After the regenerating plantshave reached the stage of shoot and root development, they may betransferred to a greenhouse for further growth and testing.

Seeds on transformed plants may occasionally require embryo rescue dueto cessation of seed development and premature senescence of plants. Torescue developing embryos, they are excised from surface-disinfectedseeds 10-20 days post-pollination and cultured. In one embodiment, themedia used for culture at this stage comprises MS salts, 2% sucrose, and5.5 g/l agarose. In embryo rescue, large embryos (defined as greaterthan 3 mm in length) are germinated directly on an appropriate media.Embryos smaller than that may be cultured for 1 week on media containingthe above ingredients along with 10⁻⁵ M abscisic acid and thentransferred to growth regulator-free medium for germination.

iii. Characterization

To confirm the presence of the exogenous MoMo30 DNA in the regeneratingplants, a variety of assays may be performed. Such assays include, forexample, Southern and northern blotting, and PCR; as well as detectionof MoMo30 protein products, e.g., by immunological means (ELISAs andWestern blots), by other assays for MoMo30 described in the Examplesbelow; by plant part assays, such as leaf or root assays; and also, byanalyzing the phenotype of the whole regenerated plant.

iv. DNA Integration, RNA Expression and Inheritance

Genomic DNA may be isolated from cell lines or any plant parts todetermine the presence of the exogenous MoMo30 gene through the use oftechniques well known to those skilled in the art. Note, that intactsequences will not always be present, presumably due to rearrangement ordeletion of sequences in the cell. The presence of MoMo30 DNA elementsintroduced through the methods of this application may be determined,for example, by polymerase chain reaction (PCR). Using this technique,discreet fragments of DNA can be amplified and detected by gelelectrophoresis. This type of analysis permits one to determine whethera gene is present in a stable transformant, but does not proveintegration of the introduced gene into the host cell genome. It istypically the case, however, that DNA has been integrated into thegenome of all transformants that demonstrate the presence of the genethrough PCR analysis. In addition, it is not typically possible usingPCR techniques to determine whether transformants have exogenous genesintroduced into different sites in the genome, i.e., whethertransformants are of independent origin. It is contemplated that usingPCR techniques it would be possible to clone fragments of the hostgenomic DNA adjacent to an introduced gene.

Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specificMoMo30 DNA sequences introduced into the host genome and flanking hostDNA sequences can be identified. Thus, the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition, it is possible through Southernhybridization to demonstrate the presence of introduced genes in highmolecular weight DNA, i.e., confirm that the introduced gene has beenintegrated into the host cell genome. The technique of Southernhybridization provides information that is obtained using PCR, e.g., thepresence of a gene, but also demonstrates integration into the genomeand characterizes each individual transformant.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, MoMo30 RNA will only be expressed in particularcells or tissue types and hence it will be necessary to prepare RNA foranalysis from these tissues. Reverse transcriptase PCR (RT-PCR)techniques also may be used for detection and quantitation of RNAproduced from the MoMo30 gene. By this approach it is first necessary toreverse transcribe the MoMo30 RNA into DNA, using enzymes such asreverse transcriptase, and then through the use of conventional PCRtechniques to amplify the DNA. In most instances, PCR techniques, whileuseful, will not demonstrate integrity of the RNA product. Furtherinformation about the nature of the RNA product may be obtained bynorthern blotting. This technique will demonstrate the presence of anRNA species and give information about the integrity of that RNA. Thepresence or absence of an RNA species also can be determined using dotor slot blot northern hybridizations. These techniques are modificationsof northern blotting and will only demonstrate the presence or absenceof an RNA species.

v. Gene Expression

While Southern blotting and PCR may be used to detect the MoMo30 gene,they do not provide information as to whether the corresponding proteinis being expressed. Expression may be evaluated by determiningexpression via transcript-profiling techniques such as by use of amicroarray, and by specifically identifying the protein products of theintroduced genes or evaluating the phenotypic changes brought about bytheir expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may be usedas well.

Assay procedures also may be used to identify the expression of proteinsby their functionality, especially the ability of enzymes to catalyzespecific chemical reactions involving specific substrates and products.These reactions may be followed by providing and quantifying the loss ofsubstrates or the generation of products of the reactions by physical orchemical procedures. Examples are as varied as the enzyme to be analyzedand may include assays for PAT enzymatic activity by followingproduction of radiolabeled acetylated phosphinothricin fromphosphinothricin and 14C-acetyl CoA or for anthranilate synthaseactivity by following loss of fluorescence of anthranilate, to name two.

Frequently, the expression of a gene product is determined by evaluatingthe phenotypic results of its expression. These assays also may takemany forms including but not limited to analyzing changes in thechemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of genesencoding enzymes or storage proteins which change amino acid compositionand may be detected by amino acid analysis, or by enzymes which changestarch quantity which may be analyzed by near infrared reflectancespectrometry. Morphological changes may include greater stature orthicker stalks. Most often changes in response of plants or plant partsto imposed treatments are evaluated under carefully controlledconditions termed bioassays.

D. Plant Breeding

In addition to direct transformation of a particular plant genotype witha MoMo30 expression construct of the current application, transgenicplants may be made by crossing a plant having MoMo30 expressionconstruct of the present application to a second plant lacking MoMo30sequences. For example, a selected CT biosynthesis gene can beintroduced into a particular plant variety by crossing, without the needfor ever directly transforming a plant of that given variety. Therefore,the current application not only encompasses a plant directlytransformed or regenerated from cells which have been transformed inaccordance with the current application, but also the progeny of suchplants. As used herein the term “progeny” denotes the offspring of anygeneration of a parent plant prepared in accordance with theapplication, wherein the progeny comprises a selected DNA constructprepared in accordance with the application. “Crossing” a plant toprovide a plant line having one or more added transgenes relative to astarting plant line, as disclosed herein, is defined as the techniquesthat result in a transgene of the application being introduced into aplant line by crossing a starting line with a donor plant line thatcomprises a transgene of the application.

In some embodiments, a method for crossing a plant to provide a plantline expressing MoMo30 comprised the steps of: (a) plant seeds of thefirst (starting line) and second (donor plant line that comprises atransgene of the application) parent plants; (b) grow the seeds of thefirst and second parent plants into plants that bear flowers; (c)pollinate a flower from the first parent plant with pollen from thesecond parent plant; and (d) harvest seeds produced on the parent plantbearing the fertilized flower.

In other embodiments, a method for backcrossing a plant expressingMoMo30 for the purpose of transferring a MoMo30 sequence from a plant ofa first genotype to a plant of a second genotype comprising the steps of(a) crossing a plant of a first genotype containing a desired gene, DNAsequence or element to a plant of a second genotype lacking the desiredgene, DNA sequence or element; (b) selecting one or more progeny plantcontaining the desired gene, DNA sequence or element; (c) crossing theprogeny plant to a plant of the second genotype; and (d) repeating steps(b) and (c) to transfer the MoMo30 sequence from a plant of a firstgenotype to a plant of a second genotype.

Introgression of a DNA element into a plant genotype is defined as theresult of the process of backcross conversion. A plant genotype intowhich a DNA sequence has been introgressed may be referred to as abackcross converted genotype, line, inbred, or hybrid. Similarly a plantgenotype lacking the desired DNA sequence may be referred to as anunconverted genotype, line, inbred, or hybrid.

The present application is further illustrated by the following examplesthat should not be construed as limiting. The contents of allreferences, patents, and published patent applications cited throughoutthis application, as well as the Figures and Tables, are incorporatedherein by reference in their entirety.

EXAMPLES Example 1. Materials and Methods

1. Plant Extracts.

One hundred grams of dried leaves from Momordica balsamina wereextracted in 1 L of distilled water overnight at 4° C. The liquidextract was separated from solid material through centrifugation at4000×g for 30 min. at 4° C. The resulting supernatant was filteredthrough Whatman filter paper (Cat #3030). The extract wasfilter-sterilized by passing it through a 0.45-micron filter (CelltreatCat #229703) and was kept frozen at −80° C. prior to lyophilizationovernight.

2. Isolation of MoMo30

The lyophilized powder was dissolved in nuclease-free water (InvitrogenCat # AM9938) to create a 15 mg/mL solution. The solution was passedthrough 30 kD molecular weight cutoff filter at 4000×g for at least 10minutes (Amicon ultra-15 cat # UFC903024) to remove low molecular weightcontaminants. Once a retentate of 1 to 1.5 mL was obtained, the solutionwas passed through a 0.22-micron syringe filter (Celltreate Cat #229747)and stored at 4° C. (−20° C. for long term storage). The retentatecontained one protein MoMo30 that was >95% pure as determined bySDS-PAGE and stained with Coomassie Brilliant Blue R-250 (Bio-Rad Cat#161-0400).

3. Multinuclear Activation of an Indicator (MAGI) Assay for Infectivity.

MAGI cell assays for infectivity were done as previously described (KhanM. et al. (2001) J Virol. 75:12081-7; Raymond A D et al. (2011) AIDS ResHum Retroviruses 27:167-178). MAGI cells (AIDS reagent program cat #U373) were grown to 90% confluence. Cells were infected with 1 ng of p24equivalent of HIV-1_(NL4-3) (AIDS reagent program cat #114). Infectedcells were fixed and identified by cells exhibiting the development ofblue color. Before staining, Cells were fixed using 1% Formaldehyde(F-79-500 Fisher Chemicals) and 0.2% Glutaraldehyde (F-02957-1 FisherScientific) in PBS. Staining solution was prepared to contain (14.25 mlPBS, 300 μl 0.2M potassium ferrocyanide, 300 μl 0.2M potassiumferricyanide, 15 μl 2M MgCl₂ and 150 μl X-gal stock (40 mg/ml in DMSO).Two ml solution was added to each well and incubated at 37° C. for 50min. Cells were washed twice with PBS and counted using lightmicroscopy.

4. Determination of the Effect of MoMo30 on Infectivity.

A MoMo30 dose-response curve was carried out using MoMo30 concentrationsfrom 1 to 100 nM. The IC50 of MoMo30 was determined by curve fittingusing the Hill equation and determined using the Dr. Fit program (DiVeroli G Y et al. (2015) Scientific Reports 5:14701. For comparison, thecommercially available fusion inhibitor Enfurvirtide (Sigma. SML0934).Briefly, MAGI cells were infected with 1 ng HIV-1NL4-3, Momo30+1 ngHIV-1NL4-3 and Enfuvirtide+1 ng HIV-1NL4-3 at different concentrationsof MoMo30 and Enfuvirtide. After 48 hrs cells were fixed and stained.

5. Detection of MoMo30 in Serum.

To determine if the ingestion of plant extracts resulted in detectablelevels of MoMo30 in the blood, two Rhesus macaques were given plantextracts using a scaled dosage to that typically given to humans. Themacaques were given the plant extracts with food. Two grams of plant wasgiven twice a day for a period of six months. Blood samples were takenat 0 days up to 183 days. Plasma was tested by SDS-PAGE and Westernblot.

6. MTT Assay of MoMo30.

To determine if MoMo30 exhibits significant cellular toxicity attherapeutic levels, HEK 293 cells were exposed to concentrations ofMoMo30 from 1 to 1000 nM and a mitochondrial toxicity test (MTT; SigmaCat # CGD-1) was performed according to the manufacturer'srecommendations. Percent viability was determined by comparison to anuntreated control.

7. Stability Studies on MoMo30 and its Complex to Gp120.

To determine the stability of MoMo30 and its attachment to HIV-1, stocksolutions of MoMo30 (4 ng/ml and 40 ng/ml) were subjected totemperatures from 25° C. to 120° C. for 30 min. After heating, thesolution was mixed with 1 ng of HIV-1_(NL4-3) and was added to a MAGIcell assay, and blue cells were counted. In a separate comparison study,1 ng of HIV-1_(NL4-3) samples were mixed with a stock solution of MoMo30(400 ng/ml), incubated at 4° C., followed by collection of aliquots attime intervals from 5 min to 3 days, and centrifuged at 125,000 gthrough 20% sucrose cushion to remove free MoMo30 and then tested forinfectivity by MAGI cell assay.

8. N-Terminal Sequencing of MoMo30

Edman degradation was performed in two labs (Biosynthesis, Lewisville,Tex., and Creative Proteomics, New York, N.Y.). The analysis wasperformed on an ABI Procise 494HT (Thermo Fisher). The proceduredetermines the N-terminal amino acid sequence of proteins and peptidesby the Edman degradation chemistry.

9. RNAseq to Determine the MoMo30 Gene Sequence.

RNAseq (GeneWiz) was used to determine the gene sequence of MoMo30.Total RNA was isolated from M. balsamina leaves by the Trizol method (˜4μg). RNAseq was done on the Illumina platform and the de novotranscriptome was assembled using Trinity software. The mRNAcorresponding to the MoMo30 protein was determined by searching for theN-terminal sequence as determined by Edman degradation. Once the DNAsequence had been determined, the MoMo30 coding sequence was synthesized(Genscript) and cloned into the pGen-lenti vector which contains both aT7 promoter and a CMV promoter for expression in mammalian cells.

10. Software for Gene Assembly and Translation.

BLAST searches were done at the national center for biotechnologyinformation (NCBI) web site. Comparisons of homology to various proteinsand DNA sequences were done in SnapGene 6.0.2. Prediction of secondarystructure was done at the Phyre2 structure prediction web portal (KelleyL A (2015) Nature Protocols 10:845-858).

11. Coupled Transcription/Translation of MoMo30 Gene.

The cloned version of the synthesized gene was expressed in the wheatgerm coupled transcription/translation system (TNT T7 coupled Wheat GermExtract System Promega Cat # L4140) according to the manufacturer'srecommendations. Ten μl of product was resolved on a 4-20% SDS-PAGE geland a Western blot was done using antibody to the N-terminal peptide ofMoMo30. Ten μl of product was also tested in a MAGI cell infectivityassay to determine any antiviral effect. Since the pGen-lenti vectoralso contained a CMV promoter, HEK293 cells were transfected with theplasmid using Lipofectamine 3000 (ThermoFisher Scientific Cat #L3000008) following the manufacturers protocol and grown for 48 hrs.Conditioned medium was harvested, and cells were collected and lysed byusing (Pierce RIPA buffer Cat #89901). Ten μl of cell pellet and 20 μlof conditioned medium was resolved on a 4-20% SDS-PAGE gel and blottedwith N-terminal antibody to MoMo30. 100 μl of conditioned medium wasused in a MAGI cell assay for evaluating antiviral effects.

12. Fluorescent Gp120 Binding Assay. P

Purified gp120 (ImmunoDX cat #1001-F) was added to a suspension of1×10⁶/ml Jurkat cells (AIDS reagent program ARP-177 E6-1 clone) allowedto interact for 2 h at 37° C. Free gp120 was removed by centrifugation,and the cells were resuspended in PBS. A portion was stained withHoechst stain, and the cells were viewed under a fluorescent microscopeusing a neutral density filter, a blue filter, and a FITC filter.

13. Surface Plasmon Resonance (Biacore).

Surface plasmon resonance was done at the Biacore Molecular InteractionShared Resource at Georgetown University. A Biacore T200 was used with aCM5 chip at 25° C. Purified gp120-IIIB (Immuno Dx, 1 mg/ml) in 1 mMsodium acetate buffer at pH 5.5 was used as a ligand to immobilize ontoFC2, FC3, and FC4 to the levels of 8850 RU, 283RU, and 2980RU,respectively. Standard amine coupling chemistry was used. HBS-P (10 mMHepes, pH 7.4, 150 mM NaCl, 0.05% v/v surfactant P20) was used as theimmobilization running buffer. Overnight kinetics were performed forMoMo30 binding to the ligand. Injected compound concentrations were 102μM to 0.1 μM. Three 15 second pulses of 1:250 H₃PO₄ (v/v, ddH₂O: H₃PO₄)were injected to regenerate the chip surface. All analyses were done intriplicate. The sensorgrams were obtained from overnight kinetics using1:1 model fitting. In some experiments, gp120 was pre-treated withPNGase F (removes N-glycans) for 30 min at 50° C. before linkage to thechip surface. A control reaction was done with buffer alone. Threeindependent assays were done, each in triplicate.

14. MoMo30 Inhibition in the Presence of Mannose.

To determine the effect of mannose on the action of MoMo30, infectivityassays were carried out with 2 nM of MoMo30 in the presence of D-Mannose(Sigma cat # M6020) at concentrations from 0.002 to 2 nM, incubated atroom temperature for five minutes, and added to MAGI cells fordetermination of infectivity inhibition by the MAGI cell assay.

15. 2G12 Antibody Blot for Changes in Gp120 Glycosylation Pattern.

The 2G12 antibody has been previously described (Scanlan C N et al.,(2002) J. Virol. 76:7306-7321). This antibody recognizes a cluster ofaspartate residues (N295, 332, 339, 386, 392) on the surface of gp120.The most likely epitope is likely to be N295 and 332. HIV-1NL4-3 wasproduced by infecting 5×10⁶ Jurkat cells (AIDS reagent program ARP-177E6-1 Clone) with 300 ng HIV-1NL4-3 (AIDS reagent program cat #114) inpresence and absence of sufficient MoMo30 (60 nM) to reduce replicationby >95%. Samples were collected 5, 8 and 12 days after infection andevaluated by Western blot using the 2G12 antibody to determine theeffect of MoMo30 on this cluster of glycans.

16. Immunoblotting.

A rabbit antibody was produced (Genescript) from a 15-amino acid peptidewith the N-terminal sequence of MoMo30. The antibody (at a dilution of1:2000) was used to perform an immunoblot on purified protein resolvedon a 4-20% SDS PAGE gel. Then were transferred to 0.2 μm Nitrocellulosemembrane using Bio-Rad Trans-Blot Turbo for 20 min and blocked with 0.5%skim milk made in Tris buffer saline with 0.1% tween 20 (TBST) for 1 hand after that membrane was incubated with primary antibody 1:2000 inTBST overnight at 4° C., Three washes of ten minutes with TBST and awash with distilled water between each wash. After which secondaryantibody (G.E. Healthcare goat anti-rabbit Cat # NA934V) was added at adilution of 1:25000 containing precision protein StrepTectin-HRP(Bio-Rad Cat #1610380) 1:10,000 and allowed to incubate for 1 hour atroom temperature. After this, the membrane was washed three times aspreviously, and chemiluminescent substrate (SuperSignal West FemtoThermo Scientific Cat #34096) was added and incubated for 5 min. Theblot was visualized by a chemiluminescent imager (ThermaFisher iBright1500). In some cases, the mouse monoclonal antibody 2G12 (AIDS reagentprogram; cat #1476) or a mouse anti-p24 antibody (AIDS reagent program;cat #6457) were used at a dilution of 1:1000 and used a mouse secondaryantibody at a dilution of 1:25000.

Example 2. Preparation and Inhibitory Activity by the MoMo30 Protein

The present application is directed to compositions and methodscomprising a 30 kDa MoMo30 product from Momordica balsamina plants. Thefunctional activity of MoMo30 protein was originally assayed fromMomordica balsamina plant extracts. In one non-limiting embodiment, themethod comprises the steps of: (a) extracting dried M. balsamina leavesin water to form an aqueous extract; (b) removing solid material fromthe aqueous extract by centrifugation to form a supernatant; and (c)filter sterilizing the supernatant. The aqueous extract therefrom can befurther frozen at −80° C. prior to lyophilization overnight.

As further described below, MoMo30-containing extracts or purifiedproteins were tested for functional activity by testing their ability toinhibit infectivity by the MAGI cell infectivity assay (or “indicatorassay”) described above. The MAGI cell infectivity assay involves theuse of genetically modified CD4-expressing HeLa cell line (MAGI)containing an HIV LTR-driven cassette placed upstream of the E. coliβ-gal encoded reporter gene (HeLa-CD4-LTR-β-gal). Cells infected by HIVturn blue and can be counted under a microscope. In the figures thatfollow, the results depict inhibition of infectivity as a function ofblue cells per nanogram of HIV-1 p24 capsid protein. In some cases, WTHIV-1_(NL4-3) was used to allow multiple rounds of infectivity. By usingHIV-1NL4-3 with a deleted env gene and then supplying env in trans, theassay can test a single round of infectivity.

Example 3. Momordica balsamina has Anti-HIV-1 Activity

Water-soluble M. balsamina extracts were tested for their ability toinhibit HIV-1NL4-3 infection in a MAGI cell assay. More particularly,cell extracts were prepared and various amounts of extract (20 μg, 50μg, 100 μg, 200 μg, 500 μg, and 1000 μg of total dry weight permilliliter of water) were mixed with 1 ng of HIV-1NL4-3 and tested fortheir ability to inhibit HIV-1NL4-3 infection in a MAGI cell reportersystem (Khan M. et al. (2001) J Virol. 75:12081-7; Raymond A D et al.(2011) AIDS Res Hum Retroviruses 27:167-178). The results of this assayshowed a dose-dependent reduction in the number of blue cells(indicating reduced infectivity), reaching 100% at 1000 μg/ml of extract(FIG. 2A). Moreover, there were no indications of cell toxicity. Thesedata indicated that the plant extracts contained one or more antiviralagents.

As a follow-up, various concentrations of purified MoMo30 protein from 1to 1000 nM were tested in a MAGI cell assay to determine the IC50 ofMoMo30-HIV binding (FIG. 2B). Similar to the results in FIG. 2A,purified protein was able to inhibit HIV-1NL4-3 in a dose-dependentfashion. The IC50 of the protein was determined by curve fitting usingthe Dr. Fit program. As shown in FIG. 2B, the IC50 was determined to be2.8 nM. The plot was curve-fit from triplicate measurements of twoindependently isolate purified protein preparations (top curve). Forcomparison, triplicate sample analysis of the commercially available HIVfusion inhibitor Enfuvirtide was conducted in parallel (FIG. 2B, bottomcurve). The IC50 of Enfuvirtide was determined to be 44 nM, consistentwith the previously published value of 26 nM (He Y et al. (2008) Proc.Natl. Acad. Sci. USA, 105:16332-16337).

Example 4. Physical and Chemical Properties of MoMo30

To further characterize the chemical nature of MoMo30, variousextraction conditions were applied to preparation of the evaluateextraction of MoMo30 from Momordica balsamina plant extracts.Accordingly, M. balsamina leaves were directly extracted in water,isopropyl alcohol (IPA), tetrahydrofuran (THF), acetylnitrile (ACN),ethanol (EtOH), and acetone (FIG. 3A). These extracts were evaluated fortheir ability to inhibit HIV infection of MAGI indicator cells, wherelower bars indicate greater anti-viral activity). As shown in FIG. 3A,the active agent can be extracted using water or acetylnitrile. Thissuggests that MoMo30 agent has both hydrophilic and hydrophobicproperties, which is characteristic of larger molecules, such asproteins. The water-soluble nature of MoMo30 was further confirmed whentesting the aqueous and organic phases obtained following extraction ofM. balsamina leaves with ethyl acetate, chloroform, hexane and diethylether (FIG. 3B).

Example 5. The Antiviral Agent of Plant Extracts is a 30 kDa Protein

To determine the relative size of the active agent, extracts were passedthrough a series of molecular weight cutoff filters ranging from 3 to100 kDa. It was determined that the antiviral activity of the extractwas retained by most of the filters (see FIG. 4A). Only at the 100 kDacutoff did more activity flow through the filter than was retained (FIG.4A). This observation suggested that the active agent was likely a largemolecule such as a protein. The extract products retained on the 30 kDacutoff filter were electrophoresed on a 4-20% SDS-PAGE gel and a singleband of approximately 30 kDa in size was detected (see FIG. 4B,Purified). Surprisingly, no other major bands were detected on theSDS-PAGE gel. Because this protein was isolated from a Momordica plantand was 30 kDa in size, the antiviral protein is referred to herein as“MoMo30”. Molecular weight cutoff filters were henceforth used toseparate MoMo30 from lower molecular weight contaminants and concentratethe protein.

Example 6. MoMo30 Stability and HIV Binding

The heat stability of MoMo30 was investigated by testing the ability ofMoMo30 to inhibit HIV infectivity by the MAGI assay after incubating theprotein at temperatures from 25° C. to 120° C. (autoclaving). Theactivity of MoMo30 was tested at concentrations of 40 ng/ml (top line)and 4 ng/ml (bottom line) over a range from 25° C. to 120° C. As shownin FIG. 4B, the percent infectivity of the purified protein remainedunchanged over the broad temperature range tested.

To determine the stability of MoMo30 complexes formed with HIV-1, 100ng/ml of MoMo30 was incubated with 1 ng of a p24 equivalent ofHIV-_(1NL4-3) virus. The complex of virus plus MoMo30 was thencentrifuged through a 30% sucrose cushion at 125,000×g to remove anyunbound MoMo30. The sucrose pellet containing virus plus bound MoMo30was then tested for infectivity by the MAGI cell assay at time intervalsfrom 5 min to 3 days. The results are summarized in FIG. 4C. Complexesof MoMo30 and HIV-1_(NL4-3) virus retained 100% of their antiviralactivity for at least 72 h suggesting that once formed, the complex ofMoMo30 and virus remains stable.

Example 7. Detection of MoMo30 with Anti-MoMo30 Antibodies

To provide a means for detection and immunopurification of MoMo30protein from cells or cell extracts, a rabbit polyclonal antibodydirected against the N-terminal amino acids of MoMo30 was generated. TheN-terminal sequence of the first 15 amino acids was determined by Edmandegradation (Creative Proteomics) to be GPIVTYWGQNVXEGEL (SEQ ID NO:16). From this peptide, a rabbit antibody made that was used to performWestern analysis to confirm that the original 30 kDa band seen in theSDS-PAGE gel was the same protein submitted for Edman degradation (seeFIG. 5A, W.B). FIG. 5B shows that in a MAGI assay, the anti-MoMo30antibody blocked the ability of MoMo30 to inhibit HIV-1 infection in adose-dependent manner (from 0.5 μg to 5.0 μg).

Example 8. Identification of the MoMo30 Gene

To identify the MoMo30 gene in Momordica balsamina, the followingprotocol was carried out: (1) isolate total plant RNA from Momordicabalsamina leaves; (2) submit RNA for RNAseq de novo transcriptomeanalysis (GeneWiz); (3) assemble reads in Trinity 2.5 software; (4)search for open reading frames (EMBOSS); (5) translate into proteinsequences (Diamond BLASTx annotation); and (6) search protein sequencesfor hevamine-related sequence motifs.

The Diamond BLAST search of open reading frames (ORFs) identified aHevamine A-like sequence translated from the RNAseq data having stronghomology (but not identical) to the N-terminal amino acid sequence(i.e., SEQ ID NO: 16) of MoMo30. The complete MoMo30 nucleotide codingsequence assembled from RNAseq reads is shown in FIG. 6 (SEQ ID NO: 1).A nucleic acid database search of the MoMo30 nucleotide coding sequencefound the MoMo30 sequence to be 93% identical to the hevamine A-likegene from M. chantaria (NCBI Reference Sequence: XM_022291555.1; FIG. 6, SEQ ID NO: 5) and 26% identity to the M. charantia MAP30 protein.Residues that are different are shaded.

A translation of the MoMo30 nucleotide coding sequence is shown in FIG.7 , panels A and B. An alignment of the MoMo30 amino acid codingsequence in SEQ ID NO: 3 with the translation product of the Momordicacharantia hevamine A-related nucleotide sequence in FIG. 6 (viaSnapGene) shows 91% identity at the protein level. Secondary structurepredictions (by Phyre2 website) of the M. balsamina MoMo30 and the M.charantia hevamine A-like ORF are shown in FIG. 7 , panel A. Thepredicted secondary structure shows strong homology to a TIM β-barrel (astructure that is commonly found in Hevamine A-like proteins (Wierenga RK, (2001) FEBS Letters 492:193-198. The TIM structure is reported to bea very heat stable conformation (Romero-Romero S et al., (2021) J. Mol.Biol., 433:167153-167153), which is consistent with the profound heatstability of MoMo30 shown in FIG. 4B.

Hevamines are members of several families of plant chitinases andlysozymes that are important for plant defense against pathogenicbacteria and fungi and belong to the family 18 glycosyl hydrolases.Hevamines are known to hydrolyze linear polysaccharide chains of chitinand peptidoglycan. The MoMo30 protein was resistant to proteolysis bymost proteases, including trypsin, which is used in most liquidchromatography with tandem mass spectrometry strategies. However, theMoMo30 protein was found to be sensitive to proteolysis by subtilisin(data not shown). Like other chitinases, MoMo30 was found to exhibitchitinase activity (data not shown).

Further, as shown in FIG. 6 , the MoMo30 sequence of SEQ ID NO: 1 has asignal peptide-encoding sequence having an amino acid sequence presentin amino acid residues 1-31 in SEQ ID NO: 7, panel A, which is removedin the secreted mature protein. FIG. 7 , panel B shows the amino acidsequence of the mature MoMo30 protein (SEQ ID NO: 4) in secreted formhaving a predicted molecular weight of 30.9 kDa. The nucleotide sequencecorresponding to the secreted form of MoMo30 is set forth in SEQ ID NO:2.

FIG. 8 shows an alignment of two conserved regions from the MoMo30protein relative to other hevamine A-related proteins comprising theamino acid sequences set forth in SEQ ID NOs: 7-15.

Example 9. In Vitro Transcription/Translation of the Hevamine A-LikeMoMo30 Gene Produces an Antiviral Effect

The MoMo30 coding sequence derived from the RNAseq data was synthesized(Genscript) and cloned into the pGen-lenti vector expression plasmid. Invitro coupled transcription and translation was carried out with theexpression plasmid using the TNT wheat germ extract system (FIG. 9 ,panel A). A MAGI assay was performed using the reaction product toconfirm that the product exhibits an antiviral effect. The synthesizedproduct (FIG. 9 , panel A) was able to inhibit HIV-1 similar to purifiedMoMo30 (FIG. 9 , panel B). The product was also reactive with theN-terminal MoMo30 antibody (FIG. 9 , panel C). Western blot analysisrevealed an ˜30 kDa protein in the supernatant, suggesting that theprotein (synthesized with the native signal peptide sequence) wassecreted, and the signal peptide was cleaved. Cell-free supernatants ofHEK-293 cells transfected with the MoMo30 expression plasmid were testedfor antiviral activity by the MAGI assay. As shown in FIG. 9 , panel D,the tissue culture supernatants were found to significantly inhibitHIV-1 infectivity. Together, these data indicate that the MoMo30 is anM. balsamina hevamine A-like protein capable of inhibiting HIV-1infection.

Example 10. MoMo30 Binds HIV-1 gp120 and Blocks its Binding to JurkatCells

To further characterize the antiviral properties of the 30 kDa hevamineA-related protein, a M. balsamina plant extract was incubated withpurified HIV-1 gp120 loaded on a non-denaturing polyacrylamide gel. Theresults of this analysis showed that the 30 kDa protein in the plantextract binds HIV-1 gp120 inducing a band-shift as shown in FIG. 10A.Further, this interaction was not disrupted by boiling or denaturingconditions of the gel (data not shown).

To further confirm this binding in the context of live cells, a blockingassay was carried out to determine the stage of viral replicationinhibited by MoMo30. To assay attachment of gp120 to susceptible cells,purified FITC labeled gp120 was mixed with Jurkat cells. FITC labeledgp120 binds the cell surface to make it visible. In the absence ofMoMo30, the purified gp120 can attach to CD4 on the surface of Jurkatcells (see FIG. 12 , panels A to C). A stock solution of 200 nM ofMoMo30 (sufficient to completely inhibit 1 ng of virus) waspre-incubated with the gp120 before adding the Jurkat suspension.Treatment with MoMo30 blocked the interaction of gp120 with Jurkat cells(compare FIG. 12 , panel A to panel D). This finding is consistent withMoMo30 blocking the initial step in replication by binding to gp120 andblocking entry.

Example 11. MoMo30 Binds to the Glycan Residues of Gp120

To further characterize the interaction of MoMo30 with gp120, surfaceplasmon resonance (Biacore) experiments were carried out. Purified gp120was bound to a chip surface, and MoMo30 at concentrations from 1 to 100nM was allowed to flow across the surface, allowing the binding to befurther characterized. Increasing concentrations of MoMo30 showedproportional reflectance increases of the chip (FIG. 13A). Threeindependent measurements in triplicate gave a kd of 2.42×10-3, K.D. of6.0 μM and ka of 400.5 l/Ms. The binding profile suggests that there isa fast on rate and a biphasic off rate. The initial dissociation israpid, followed by a very slow dissociation.

To further characterize the binding of MoMo30 to gp120, purified gp120was pre-treated with PNGase F. PNGase F is an amidase that works bycleaving between the innermost GlcNAc and asparagine residues of highmannose, hybrid, and complex oligosaccharides from N-linkedglycoproteins and glycopeptides, resulting in a deaminated protein orpeptide and a free glycan. Following treatment with PNGase F, a dramaticdecrease in binding to the chip surface was observed (see FIG. 13B), theloss of sugar residues produced a decrease in reflectance units (RU),reflecting a decrease in MoMo30 binding to gp120, suggesting that MoMo30binds to glycan residues in gp120.

Example 12. Effect of Mannose Monosaccharide and Glycan Specific mAbs onInhibition of Infection by MoMo30

The glycans on gp120 comprise a mixture of high mannose glycans andcomplex-type mannose glycans. To determine the effect of adding mannosein the context of MoMo30 binding to HIV-1NL4-3, HIV-1NL4-3 was incubatedwith MoMo30 (2 nM) in the presence of mannose concentrations from 0 to 2nM (FIG. 14A). The results of this experiment showed that inhibition ofHIV by MoMo30 was entirely abolished by the presence of mannose at aconcentration of 2 nM, which is a 1:1 molar ratio with MoMo30, therebysuggesting a single binding site in gp120 (FIG. 14A).

The monoclonal antibody 2G12 has been previously shown to react withspecific high mannose glycans on the surface of gp120 (Punja Z K et al.(1993) J Nematol 25:526-40; Sahai A S et al. (1993) FEMS MicrobiologyReviews 11:317-338), most likely N295 and N332 (Scanlan C N et al.,(2002) J. Virol. 76:7306-7321). To determine the effect of MoMo30 onreactivity with the antibody 2G12, Jurkat cells were infected withHIV-1NL4-3 in the presence or absence of 1 nM MoMo30. Virus washarvested on days 5, 8, and 12 after initial exposure and the resultingparticles were subjected to immunoblot analysis using the mAb 2G12 (FIG.14B). The results of this experiment showed that exposing HIV-1 toMoMo30 appears to reduce reactivity over time with the glycan specificmAb 2G12. This is consistent with the observation that MoMo30 binds toglycan residues on the surface of gp120 and supports the conclusion thatcarbohydrate residues become inaccessible as a result of the binding.

Example 13. MoMo30 Binds to Other Viruses

To determine whether the antimicrobial properties of the active agent inthe plant extracts are specific for HIV or could be extended to otherviruses as a broad-spectrum antimicrobial agent, a MAGI cell infectivityassay was performed in simian immunodeficiency virus (SIV) infectedcells, the results of which are shown in FIG. 15 . Additionally, EBOVassays were carried out in Ebola virus infected cells treated withseveral different independently obtained MoMo30 extracts (A-E).Infectivity was determined by immunofluorescence using anti-EBOVantibodies. As shown in FIG. 16 , each of the MoMo30 extracts was shownto exhibit anti-EBOV activity in both Hela and HFF cell lines asindicated by the EC50 (concentration required to obtain a 50% effect).For all extracts, the potency values were slightly better in Hela cellsthan in HFF. The safety index (SI) was low for extracts A and C in Helacells.

As an extension of this analysis, an envelope (env) deleted HIV-1 strain(KFS) pseudotyped to contain the MuMLV envelope glycoprotein was testedfor infectivity in the absence (left) or presence (right) of MoMo30(FIG. 17 ). The results of this assay showed that an HIV-1 pseudotypedwith the aMLV envelope protein is sensitive to MoMo30 inhibition,suggesting that the antiviral properties of MoMo30 broadly extend to avariety of enveloped viruses via glycosylated surface envelopes. Takentogether, these results are consistent with the active agent havingbroad-spectrum antimicrobial properties.

Example 14. Orally Provided MoMo30 can Accumulate Stably in theBloodstream of a Non-Human Primate

To determine MoMo30 bioavailability in the bloodstream, M. balsaminaextracts of the medicinal plants were administered to two Rhesusmacaques by mouth for six months. MAGI infectivity assays and Westernblots were carried out to confirm the presence of MoMo30 in the serum oftreated animals (FIG. 18 ). Serum was tested from 0 to 183 days afteringestion. Neither of the animals exhibited inhibitory activity in theirserum prior to ingestion of the plant extract. However sera from bothpossessed significant amounts of antiviral activity by 42 dayspost-ingestion (FIG. 2C). A Western blot was carried out using theanti-MoMo30 antibody described herein. A representative Western blot ofserum from the animal is represented by the cross-hatched bars is shownas an inset to FIG. 18 . Neither animal had detectable protein presentin their serum before ingestion.

Example 15. MoMo30 is not Toxic to MAGI Cells at Therapeutic Levels

To confirm the absence of toxicity at the cellular level, the effect ofvarious MoMo30 concentrations (1 to 1000 nM) on MAGI cells wasevaluated. Specifically, a cellular viability (MTT) assay measuring acell's metabolic activity was carried out in which metabolic activity isreduced if the compound is toxic. As shown in FIG. 19 , the treatedcells showed no toxic effect at concentrations needed to inhibit HIVinfection.

Example 16. MoMo30 Causes Hemagglutination

The observation that MoMo30 appears to bind sugar groups in a range ofviral envelopes suggests that it has properties reminiscent of lectins.Inasmuch as lectins have often been found to exhibit hemagglutininactivity, it was of interest to investigate whether MoMo30 exhibitshemagglutinin activity too. FIG. 20A shows the results of this analysis.In this case, purified MoMo30 protein was tested for its ability toagglutinate sheep red blood cells (RBCs). As shown in panel A, a 30mg/ml stock solution at a dilution of 1:512 was found to causehemagglutination, consistent with lectin-like activity.

Example 17. MoMo30 Stimulates the Activation and Proliferation of TCells

Inasmuch as lectins are known to function as T cell mitogens, such asphytohemagglutinin A (PHA), it was of interest to examine whether MoMo30can stimulate the activation and proliferation of T cells. Thus, a Tcell activation assay was performed in which a fixed number of Jurkatcells was treated (left to right) with PBS (neg. control, Con), PHA(pos. control), or MoMo30 (FIG. 20B). The results of this assay showedthat MoMo30 similarly stimulates the activation and proliferation of Tcells.

Example 18. Clinical Study of HIV Patients Treated with a MoMo30 HerbalTea

To examine the therapeutic efficacy of the MoMo30 protein, HIV-infectedpatients (n=61) were orally administered a combination of Extracts A-Edaily for a period of 6 months during which no other anti-retroviralagents (ARVs) were administered. The extracts were administered in theform of an herbal tea. During this 6 month treatment period, thepatients' viral loads (FIG. 21A) and CD4+ lymphocyte counts (FIG. 21B)were monitored. The results of this analysis showed that the averageviral load was significantly reduced (FIG. 21A), while the CD4+ cellcounts increased over this same period (FIG. 21B). A follow-up done over10 years later (180 months) showed that some of the treated patients arehealthy and exhibit undetectable or extremely low HIV virus levels (FIG.21A).

Table 1 shows individual patient data evaluating viral loads and CD4+cell counts in the follow-up patients compared to healthy, uninfectedcontrol subjects, where NP=Not performed (specimen clotted) and ND=Notdetected <20 copies/ml.

TABLE 1 % CD4⁺ Patient cells/total No. CD4⁺ No. total Viral Load Numberlymphocytes cells/ml lymphocytes/ml (<20 copies) Uninfected ControlSubjects 1 26 422 1630 ND 2 53 539 1019 ND 3 23 175 762 ND 4 50 116 230ND 5 28 211 761 ND 6 21 374 1797 ND 7 32 139 434 ND 8 45 185 414 ND 9 2882 298 ND 10 13 206 1634 ND 11 37 565 1518 ND 12 49 106 215 ND 13 NP NPNP ND Control Avg 34 260 893 Follow-Up Patients 10 Years After Treatment14 21 95 444 ND 15 36 61 170 ND 16 47 73 154 ND 17 16 146 895 3360 18 2478 322 ND 19 33 969 2909  20 20 21 180 855 ND 21 32 95 294 3600 22 14110 768 ND 23 29 314 1094 ND 24 32 283 892 ND 25 29 649 2253 ND 26 13112 897 ND Patient Avg 27 243 919

The results of the follow-up study showed that the treated patientsshowed similar CD4 and viral load profiles compared to the uninfectedcontrol subjects.

FIG. 22 , panels A and B further show the results of the clinical studyabove, further documenting an increase in CD4+ lymphocytes of about 50%following 6 months of treatment with MoMo30 plant extract (FIG. 22 ,panel A), and a decrease of 60% of the patients' mean HIV viral loadsfollowing a 6-months post-treatment (FIG. 22 , panel B), and in mostcases decreased to undetectable levels after 180 months (FIG. 22 , panelB).

In FIG. 22 , panel C, a subset of the original patients (n=13) werere-tested at 180 months. The results of this analysis showed that CD4counts in most of the re-tested patients returned to near baselinelevels. In addition, viral loads in most of these re-tested patients haddecreased to undetectable (<20 copies/ml) levels at 180 monthspost-treatment.

Example 19. Analysis of Neutralizing Antibody Production

Antisera from the subset of original patients (n=13) in FIG. 22C werefurther evaluated for production of neutralizing antibodies in an HIVneutralizing antibody assay as previously described (Simek et al., J.Virol., 83(14):7337-7348, July 2009). Multiple assay controls were setup to monitor each plate in a run and to allow for comparison of runsover time. The two types of controls included: (1) a control virus paneltested with all samples (sera, plasma, antibodies, etc.) and (2) anantibody control. The control virus panel includes the neutralizationsensitive lab strain env, HIV-1_(N4-3); a less sensitive primary isolateenv, JRCSF; and a specificity control env, amphotropic murine leukemiavirus (aMLV) envelope. aMLV was used as a specificity control, becauseit is a non-HIV envelope and has not been found to be inhibitable byantibodies to HIV. Any inhibition of aMLV by plasma would be attributedto non-specific factors. The antibody control included a broadlyneutralizing HIV+ plasma present on all control assay plates.

FIG. 23 , panels A-C summarize the results of the HIV neutralizingantibody assay using antisera obtained from the 13 re-tested patients at180 months post-treatment. FIG. 23 , panels A and B show the results ofthe patient PROM050 serum being tested in a MAGI indicator cell assayfor neutralizing activity against HIV-1 pseudotyped with an HIV-1NL4-3env or an aMLV env, respectively. FIG. 23 , panel C shows a tabledepicting antibody titers from the 13 re-tested patients at 180 monthspost-treatment against each of an HIV-1 pseudotyped with an HIV-1envelope from one or 10 primary strains or one of 3 lab strains ofHIV-1, as indicated. In this case, the serum from each of these 13patients was tested against 13 different isolates of HIV-1 in 5different clades. The table in FIG. 23 , panel C summarizes reciprocaldilutions of the inhibitory dose to induce 50% reduction in replicationof virus (ID 50) as measured in a MAGI indicator cell assay. Darkershaded areas depict higher titers, while the lighter shaded areas depictlower titers.

Seven of these individuals had high titers of antibody against all ofthe isolates. Six of the patients had lower levels of antibody. All ofthe patients had significant levels of neutralizing antibodies againstthe common lab strain, HIV-1NL4-3. Not wishing to be bound by theory,the results suggest that binding of MoMo30 to glycosyl groups of gp120exerts pressure for selection of mutant viruses having fewer glycosylgroups so the virus will be less susceptible to MoMo30. Thus, viruseswith fewer sugars will be more antigenic and allow the host to mount aneutralizing ab response.

The follow up patients were tested for neutralizing ab at 180 months.More than half of them show high levels of ab that can neutralize over adozen strains of HIV-1. The pseudotyped strains were used to testdifferent primary envelope proteins. MuMLV env was used as a negativecontrol, since there was no expectation for subjects being infected withthis mouse virus. However, half of the patients also had an antibodythat could neutralize that control, suggesting a broadly cross-reactiveantibody. These results suggest that neutralizing antibodies raised inresponse to MoMo30 are responsible for the long-term control of HIVinfection.

FIG. 24 shows that the neutralizing activity of serum from two patients(PROM050 and PROM052) treated with MoMo30 is completely eliminatedfollowing 3 successive rounds of Protein A/G adsorption. Additionally,these results confirm that the inhibitory activity was solely attributedto antibodies rather than an effect caused by the use of otheranti-retroviral agents.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications andvariations. While various embodiments have been described above, itshould be understood that such disclosures have been presented by way ofexample only and are not limiting. Thus, the breadth and scope of thesubject compositions and methods should not be limited by any of theabove-described exemplary embodiments and should be defined only inaccordance with the following claims and their equivalents, which shouldbe understood to cover obvious modifications and variations which arereadily apparent to a person of ordinary skill in the art upon readingthe description. Further, the claims are intended to cover thecomponents and steps in any sequence which is effective to meet theobjectives there intended, unless the context specifically indicates thecontrary.

What is claimed is:
 1. A nutraceutical composition, comprising: anantimicrobial hevamine A-related protein comprising an amino acidsequence at least 95% identical to SEQ ID NO:3 or SEQ ID NO:4; and atleast one nutraceutically acceptable carrier.
 2. The nutraceuticalcomposition of claim 1, further comprising one or more nutraceuticalingredients selected from the group consisting of antimicrobial agents,immune-stimulating agents, anti-inflammatory agent, antioxidant agent,and combinations thereof, wherein the one or more nutraceuticalingredients comprise zinc and quercetin.
 3. The nutraceuticalcomposition of claim 1, formulated for oral, intravenous orintramuscular administration.
 4. The nutraceutical composition of claim1, wherein the composition is formulated in the form of a capsule, atablet or a lozenge.
 5. A method for preparing the nutraceuticalcomposition of claim 1, comprising the steps of: drying a plantcomprising a protein comprising an amino acid sequence at least 95%identical to SEQ ID NO:4; extracting the dried plant in an aqueousmedium; and separating the aqueous medium from solid material to form anaqueous extract, wherein the aqueous extract comprises the protein. 6.The method of claim 5, further comprising the step of purifying theprotein from the aqueous extract by immunoaffinity purification togenerate a purification product.
 7. The method of claim 6, furthercomprising the step of adding one or more nutraceutical ingredients tothe purification product, wherein the one or more nutraceuticalingredients are selected from the group consisting of antimicrobialagents, immune-stimulating agents, anti-inflammatory agent, antioxidantagent, and combinations thereof, and wherein the one or morenutraceutical ingredients comprise zinc and quercetin.
 8. The method ofclaim 5, further comprising the step of: passing the aqueous extractthrough a molecular weight cut-off filter; collecting a retentatecomprising the protein; and purifying the protein from the retentate togenerate a purification product.
 9. The method of claim 8, furthercomprising the step of adding one or more nutraceutical ingredients tothe purification product, wherein the one or more nutraceuticalingredients are selected from the group consisting of antimicrobialagents, immune-stimulating agents, anti-inflammatory agent, antioxidantagent, and combinations thereof, and wherein the one or morenutraceutical ingredients comprise zinc and quercetin.
 10. A method forpreventing or reducing symptoms of a condition in a subject, comprisingadministering to the subject an effective amount of the nutraceuticalcomposition of claim
 1. 11. The method of claim 10, wherein thecondition comprises microbial infection diseases, abnormal high energymetabolism or low energy metabolism.
 12. The method of claim 10, whereinthe higher energy metabolism is anemia, pregnancy, growth, exercise,cancers, recovery from surgical and other injuries.
 13. The method ofclaim 12, wherein the low energy metabolism comprises malnutrition,anorexia, or aging.
 14. The method of claim 10, wherein the microbialinfection is caused by a virus, wherein the virus is HIV, influenza Type1 virus, SARS-CoV-2, SARS-CoV-2 or MERS-CoV.
 15. The method of claim 10,wherein nutraceutical composition is administered to the subject orally,intravenously or intramuscularly.
 16. A method of preventing or treatinga microbial plant infection, comprising: applying an effective amount ofa composition, either pre- or post-infection, to a plant, plant part, ormedia in which a plant is growing, wherein the composition comprises anantimicrobial hevamine A-related protein comprising an amino acidsequence at least 95% identical to SEQ ID NO:3 or SEQ ID NO:4, whereinthe plant part is selected from the group consisting of leaves, roots,stems, fruit, seeds, tubers, bulbs, flowers, pods, stems, shoots, andcombinations thereof.
 17. The method of claim 16, wherein thecomposition is applied by spraying a liquid or powder formulation to theplant, plant part, or a medium in which the plant is growing.
 18. Atransgenic plant, transgenic plant part, or transgenic plant cell,comprising: a stably integrated DNA expression construct comprising: apolynucleotide comprising a nucleotide sequence at least 95% identicalto SEQ ID NO:1 or SEQ ID NO:2, or a polynucleotide encoding a proteincomprising an amino acid sequence at least 95% identical to SEQ ID NO:3or SEQ ID NO:4, wherein the transgenic plant, transgenic plant part, ortransgenic plant cell exhibits increased resistance to at least onebacterial, fungal or viral infection as compared to a control plant,plant part, or plant cell lacking the DNA expression construct.
 19. Thetransgenic plant, transgenic plant part, or transgenic plant cell ofclaim 18, wherein the transgenic plant, transgenic plant part, ortransgenic plant cell is derived from a crop plant selected from thegroup consisting of wheat, corn, rice, barley, cotton, canola, alfalfa,sugarbeet, potato and tomato.
 20. A method of producing a transgenicplant, comprising the steps of: (a) stably transforming into a hostplant a recombinant DNA expression construct comprising: apolynucleotide comprising a nucleotide sequence at least 95% identicalto SEQ ID NO:1 or SEQ ID NO:2, or a polynucleotide encoding a proteincomprising an amino acid sequence at least 95% identical to SEQ ID NO:3or SEQ ID NO:4; and (b) isolating a transgenic plant expressing thestably transformed polynucleotide in an amount sufficient to provideincreased resistance to a fungal, bacterial or viral infection ascompared to a control plant lacking the polynucleotide under the samecondition, wherein the host plant is a crop plant selected from thegroup consisting of wheat, corn, rice, barley, cotton, canola, alfalfa,sugarbeet, potato and tomato.